Engineering of multi-carbon substrate utilization pathways in methanotrophic bacteria

ABSTRACT

The present disclosure relates to genetically engineered methanotrophic bacteria with the capability of growing on a multi-carbon substrate as a primary or sole carbon source and methods for growing methanotrophic bacteria on a multi-carbon substrate.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 200206_402USPC_SEQUENCE_LISTING.txt. The textfile is 399 KB, was created on Apr. 24, 2015, and is being submittedelectronically via EFS-Web.

BACKGROUND

Technical Field

The present disclosure relates to genetically engineered methanotrophicbacteria capable of growing on multi-carbon substrates and methods forgrowing methanotrophic bacteria on multi-carbon substrates.

Description of the Related Art

Methanotrophic bacteria generally rely on methane as their sole carbonand energy source. The low solubility of methane is a major limitingfactor in achieving high cell density and rapid growth in methanotrophicbacteria. The slow growth of methanotrophs and their requirement formethane, a potentially explosive substrate, has hampered theirindustrial application. For many industrial applications, such aschemical catalysis or other biological transformations, it is desirableto achieve high amounts of methanotroph biomass, regardless of thecarbon source used.

In view of the limitations associated with methanotrophic bacteriagrowth, there is a need in the art for methanotrophs that can utilizealternative, preferably inexpensive, substrates as carbon and energysources. The present disclosure provides a solution by providinggenetically engineered methanotrophic bacteria that can utilizemulti-carbon substrates, including glycerol.

SUMMARY OF INVENTION

In one aspect, the present disclosure provides for recombinant obligatemethanotrophic bacteria including at least one exogenous nucleic acidencoding a multi-carbon substrate utilization pathway component, whereinthe at least one exogenous nucleic acid is expressed in a sufficientamount to permit growth of the non-naturally occurring methanotrophicbacteria on the multi-carbon substrate as a primary carbon source. Incertain embodiments, the multi-carbon substrate is a sole carbon source.In certain embodiments, the multi-carbon substrate may be glucose,acetate, lactate, arabinose, citrate, succinate, or glycerol.

In another aspect, the present disclosure provides for recombinantfacultative methanotrophic bacteria including at least one exogenousnucleic acid encoding a multi-carbon substrate utilization pathwaycomponent, wherein the multi-carbon substrate is not utilized as acarbon source by a reference facultative methanotrophic bacterium,wherein the at least one exogenous nucleic acid encoding a multi-carbonsubstrate utilization pathway component is expressed in a sufficientamount to permit growth of the recombinant facultative methanotrophicbacteria on the multi-carbon substrate as a sole carbon source. Incertain embodiments, the multi-carbon substrate may be glucose,glycerol, lactate, arabinose, acetate, succinate, or citrate.

In certain embodiments wherein the multi-carbon substrate is glucose,the recombinant methanotrophic bacteria include an exogenous nucleicacid encoding a glucose transporter.

In certain embodiments wherein the multi-carbon substrate is acetate,the recombinant methanotrophic bacteria include an exogenous nucleicacid encoding an acetate transporter. In further embodiments, thenon-naturally occurring methanotrophic bacteria are further modified tooverexpress acetyl-CoA synthase.

In certain embodiments wherein the multi-carbon substrate is lactate,the recombinant methanotrophic bacteria include an exogenous nucleicacid encoding a lactate transporter and an exogenous nucleic acidencoding a lactate dehydrogenase.

In certain embodiments wherein the multi-carbon substrate is arabinose,the recombinant methanotrophic bacteria include an exogenous nucleicacid encoding an L-arabinose isomerase, an exogenous nucleic acidencoding an L-ribulose kinase, an exogenous nucleic acid encoding anL-ribulose-5-phosphate epimerase, and an exogenous nucleic acid encodingan arabinose transporter. In further embodiments, the L-arabinoseisomerase is AraA, the L-ribulose kinase is AraB, theL-ribulose-5-phosphate epimerase is AraD, and the arabinose transporteris AraE, AraFGH, or AraP.

In certain embodiments wherein the multi-carbon substrate is citrate,the recombinant methanotrophic bacteria include an exogenous nucleicacid encoding a citrate transporter.

In certain embodiments wherein the multi-carbon substrate is succinate,the recombinant methanotrophic bacteria include an exogenous nucleicacid encoding a succinate transporter.

In certain embodiments wherein the multi-carbon substrate is glycerol,the recombinant methanotrophic bacteria include at least two exogenousnucleic acids encoding glycerol utilization pathway components. Infurther embodiments, the at least two glycerol utilization componentscomprise glycerol kinase and glycerol-3-phosphate dehydrogenase. In aspecific embodiment, the glycerol kinase is GlpK andglycerol-3-phosphate dehydrogenase is GlpD.

In further embodiments, the recombinant methanotrophic bacteria includethree exogenous nucleic acids encoding glycerol utilization pathwaycomponents. In still further embodiments, the three glycerol utilizationpathway components comprise glycerol uptake facilitator, glycerolkinase, and glycerol-3-phosphate dehydrogenase. In a specificembodiment, the glycerol uptake facilitator is GlpF, the glycerol kinaseis GlpK, and the glycerol-3-phosphate dehydrogenase is GlpD.

In certain embodiments, the recombinant obligate methanotrophic bacteriaare Methylomonas, Methylobacter, Methylococcus, Methylosinus,Methylomicrobium, or Methanomonas. In further embodiments, therecombinant obligate methanotrophic bacteria are Methylosinustrichosporium strain OB3b, Methylococcus capsulatus Bath strain,Methylomonas methanica 16A strain, Methylosinus trichosporium (NRRLB-11,196), Methylosinus sporium (NRRL B-11,197), Methylocystis parvus(NRRL B-11,198), Methylomonas methanica (NRRL B-11,199), Methylomonasalbus (NRRL B-11,200), Methylobacter capsulatus (NRRL B-11,201),Methylomonas sp AJ-3670 (FERM P-2400), Methylacidiphilum infernorum, orMethylomicrobium alcaliphilum 20Z.

In certain embodiments, the recombinant facultative methanotrophicbacteria are Methylocella silvestris, Methylocella palustris,Methylocella tundrae, Methylocystis daltona strain SB2, Methylocystisbryophila, Methylocapsa aurea KYG, or Methylobacterium organophilum(ATCC 27,886).

In certain embodiments, at least one exogenous nucleic acid encoding amulti-carbon substrate utilization pathway component is codon optimizedfor high expression in methanotrophic bacteria.

Additionally, the present disclosure provides methods for growingmethanotrophic bacteria, comprising culturing the recombinant obligatemethanotrophic bacteria according to any of the embodiments providedherein in the presence of a multi-carbon substrate, wherein themulti-carbon substrate is used as a primary carbon source by therecombinant obligate methanotrophic bacteria. In certain embodiments,the present disclosure provides methods for growing methanotrophicbacteria, comprising culturing the recombinant faculatativemethanotrophic bacteria according to any of the embodiments providedherein in the presence of a multi-carbon substrate, wherein themulti-carbon substrate is used as a sole carbon source by therecombinant faculatative methanotrophic bacteria.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows an exemplary glycerol utilization pathway (respiratory) fora genetically modified methanotrophic bacterium. Glycerol crosses thecell membrane (e.g., via a glycerol uptake facilitator such as GlpF),where it is phosphorylated by a glycerol kinase (GK) (e.g., GlpK) toform glycerol-3-phosphate (G3P), which is then oxidized byglycerol-3-phosphate dehydrogenase (e.g., GlpD) to dihydroxyacetonephosphate (DHAP), which is then isomerized by triose phosphate isomeraseand may then enter endogenous sugar metabolism or gluconeogenesispathways.

DETAILED DESCRIPTION

Prior to setting forth this disclosure in more detail, it may be helpfulto an understanding thereof to provide definitions of certain terms tobe used herein. Additional definitions are set forth throughout thisdisclosure.

In the present description, the term “about” means±20% of the indicatedrange, value, or structure, unless otherwise indicated. The term“consisting essentially of” limits the scope of a claim to the specifiedmaterials or steps and those that do not materially affect the basic andnovel characteristics of the claimed invention. It should be understoodthat the terms “a” and “an” as used herein refer to “one or more” of theenumerated components. The use of the alternative (e.g., “or”) should beunderstood to mean either one, both, or any combination thereof of thealternatives. As used herein, the terms “include” and “have” are usedsynonymously, which terms and variants thereof are intended to beconstrued as non-limiting. The term “comprise” means the presence of thestated features, integers, steps, or components as referred to in theclaims, but that it does not preclude the presence or addition of one ormore other features, integers, steps, components, or groups thereof.

As used herein, the term “recombinant” or “non-natural” refers to anorganism, microorganism, cell, nucleic acid molecule, or vector that hasat least one genetic alternation or has been modified by theintroduction of an exogenous nucleic acid, or refers to a cell that hasbeen altered such that the expression of an endogenous nucleic acidmolecule or gene can be controlled, where such alterations ormodifications are introduced by genetic engineering. Genetic alterationsinclude, for example, modifications introducing expressible nucleic acidmolecules encoding proteins or enzymes, other nucleic acid additions,nucleic acid deletions, nucleic acid substitutions, or other functionaldisruption of the cell's genetic material. Such modifications include,for example, coding regions and functional fragments thereof forheterologous or homologous polypeptides for the referenced species.Additional modifications include, for example, non-coding regulatoryregions in which the modifications alter expression of a gene or operon.Exemplary proteins or enzymes include proteins or enzymes (i.e.,components) within a multi-carbon substrate utilization pathway (e.g., aglycerol utilization pathway). Genetic modifications to nucleic acidmolecules encoding enzymes, or functional fragments thereof, can confera biochemical reaction capability or a metabolic pathway capability tothe recombinant cell that is altered from its naturally occurring state.

As used herein, the term “host bacterium” or “host” refers to amicroorganism (e.g., methanotrophic bacterium) that has not yet beengenetically modified with the capability to utilize a multi-carbonsubstrate (e.g., glycerol) as a carbon and energy source. A hostmethanotrophic bacterium is selected for transformation with at leastone exogenous nucleic acid encoding a multi-carbon substrate utilizationpathway component to yield a recombinant methanotrophic bacterium withthe capability to utilize a multi-carbon substrate as a carbon andenergy source. A host methanotrophic bacterium may already possess othergenetic modifications conferring it with desired properties, unrelatedto the multi-carbon substrate utilization pathway disclosed herein. Forexample, a host methanotrophic bacterium may possess geneticmodifications conferring high growth, tolerance of contaminants orparticular culture conditions, ability to metabolize additional carbonsubstrates, or ability to synthesize desirable products or intermediates(e.g., propylene, crotonate, or crotonyl CoA, see, e.g., InternationalApplication Number PCT/US13/60460, incorporated herein by reference, inits entirety).

As used herein, the term “methanotrophic bacterium” refers to amethylotrophic bacterium that has the ability to oxidize methane as itssole carbon and energy source. As used herein, “methanotrophic bacteria”include “obligate methanotrophic bacteria” that can only utilize C1substrates for carbon and energy sources and “facultative methanotrophicbacteria” that are naturally able to use multi-carbon substrates, suchas acetate, pyruvate, succinate, malate, or ethanol, in addition to C1substrates as their sole carbon and energy source. Facultativemethanotrophs include some species of Methylocella, Methylocystis, andMethylocapsa (e.g., Methylocella silvestris, Methylocella palustris,Methylocella tundrae, Methylocystis daltona strain SB2, Methylocystisbryophila, and Methylocapsa aurea KYG), and Methylobacteriumorganophilum (ATCC 27,886).

As used herein, the term “reference facultative methanotrophicbacterium”, as known as “wild type facultative methanotrophic bacterium”or “parent facultative methanotrophic bacterium”, refers to afacultative methanotrophic bacterium that has not been geneticallyengineered with the capability to use an additional multi-carbonsubstrate other than its native substrates.

As used herein, the term “not utilized as a carbon source” means thatthe referenced carbon substrate cannot be used as a sole carbon sourceby the referenced bacteria.

As used herein, the term “C1 substrate” or “C1 compound” refers to anyorganic compound that lacks a carbon to carbon bond. C1 substratesinclude methane, methanol, formaldehyde, formic acid (formate), carbonmonoxide, carbon dioxide, methylated amines (e.g., methylamine,dimethylamine, trimethylamine, etc.), methylated thiols, methyl halogens(e.g., bromomethane, chloromethane, iodomethane, dichloromethane, etc.),and cyanide.

As used herein, the term “multi-carbon substrate” or “multi-carboncompound” refers to an organic compound that contains at least onecarbon to carbon bond. A multi-carbon substrate includes organic acidsand carbohydrates. Exemplary multi-carbon substrates include glucose,acetate, lactate, arabinose, citrate, succinate, and glycerol.

As used herein, the term “glucose”, also known as “D-glucose” or“dextrose”, refers to a colorless, water soluble organic compound havingthe formula C₆H₁₂O₆ and that is the D-isomer or “right-handed form” ofglucose. As used herein, glucose refers to both the open-chain form aswell as cyclic isomers (e.g., α-D-glucopyranose, β-D-glucopyranose,α-D-glucofuranose, and β-D-glucofuranose).

As used herein, the term “acetate”, also known as “ethanoate”, refers toan organic compound that is a salt or ester of acetic acid (chemicalformula CH₃CO₂H (also written as CH₃COOH)). The formula for acetateanion is CH₃CO₂ ⁻, C₂H₃O₂ ⁻, or CH₃COO⁻. Acetate may also be abbreviatedas OAc⁻ or AcO⁻.

As used herein, the term “lactate” refers to a salt or ester of lacticacid. Lactic acid, also known as 2-hydroxypropanoic acid or sarcolacticacid, is a carboxylic acid with the chemical formula C₃H₆O₃. The lactateanion has the chemical formula CH₃CH(OH)COO⁻. Lactate includes L-lactateand D-lactate optical isomers.

As used herein, the term “arabinose”, also known as “pectinose”, refersto a monosaccharide containing five carbon atoms including an aldehydefunctional group (aldopentose). Arabinose has the chemical formulaC₅H₁₀O₅. As used herein, arabinose includes L-arabinose and D-arabinose.

As used herein, the term “citrate” refers to a salt or ester of citricacid. Citric acid, also known as 2-hydroxypropane-1,2,3-tricarboxylicacid, is a weak organic acid that has the chemical formula C₆H₈O₇. Thecitrate anion has the chemical formula C₃H₅O(COO)₃ ³⁻.

As used herein, the term “succinate” refers to a salt or ester ofsuccinic acid. Succinic acid, also known as butanedioic acid orethane-1,2-dicarboxylic acid, has the chemical formula C₄H₆O₄.

As used herein, the term “glycerol”, also known as glycerine, glycerin,1,2,3-propanetriol, glyceritol, glycyl alcohol, trihydroxypropane,propanetriol, osmoglyn, or 1,2,3-trihydroxypropane, refers to atri-hydroxy sugar alcohol with the formula C₃H₈O₃. It is a colorless,odorless, viscous liquid. Glycerol is an intermediate in carbohydrateand lipid metabolism and is often used as a solvent, emollient,pharmaceutical agent, and sweetening agent. As used herein, glycerolincludes both purified glycerol, the form used in pharmaceutical, food,and cosmetic industries, and crude glycerol. Crude glycerol, or g-phase,is a heavier separate liquid phase composed mainly of glycerol that isthe by-product of biodiesel production. Crude glycerol generated byhomogeneous base-catalyzed transesterification contains approximately50-60% of glycerol, 12-16% of alkalies, especially in the form of alkalisoaps and hydroxides, 15-18% of methyl esters, 8-12% of methanol, 2-3%water, and further components. Crude glycerol also contains a variety ofelements, such as calcium, magnesium, phosphorus, or sulfur, originatingfrom the primary oil. Larger quantities of sodium or potassium are alsopresent, derived from the catalyst.

As used herein, “exogenous” means that the referenced molecule (e.g.,nucleic acid) or referenced activity (e.g., enzyme activity or membranetransport) is introduced into a host methanotrophic bacterium by geneticengineering. The molecule can be introduced, for example, byintroduction of a nucleic acid into the host genetic material such as byintegration into a host chromosome or by introduction of a nucleic acidas non-chromosomal genetic material, such as on a plasmid. When the termis used in reference to expression of an encoding nucleic acid, itrefers to introduction of the encoding nucleic acid in an expressibleform into the host methanotrophic bacterium. When used in reference toan enzymatic or protein activity, the term refers to an activity that isintroduced into the host reference bacterium. Therefore, the term“endogenous” or “native” refers to a referenced molecule or activitythat is present in the host bacterium. The term “chimeric” when used inreference to a nucleic acid refers to any nucleic acid that is notendogenous, comprising sequences that are not found together in nature.For example, a chimeric nucleic acid may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences that are derived from the samesource, but arranged in a manner different than that found in nature.The term “heterologous” refers to a molecule or activity that is derivedfrom a source other than the referenced species or strain whereas“homologous” refers to a molecule or activity derived from the hostbacterium. Accordingly, a methanotrophic bacterium comprising anexogenous nucleic acid as provided in the present disclosure can utilizeeither a heterologous or homologous nucleic acid or both.

It is understood that when more than one exogenous nucleic acid isincluded in a bacterium that the more than one exogenous nucleic acidrefers to the referenced encoding nucleic acid or protein activity, asdiscussed above. It is also understood that such more than one exogenousnucleic acid can be introduced into the host bacterium on separatenucleic acid molecules, on a polycistronic nucleic acid molecule, on asingle nucleic acid molecule encoding a fusion protein, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein, a methanotrophic bacterium canbe modified to express two or more exogenous nucleic acids encoding adesired multi-carbon substrate utilization pathway component (e.g.,glycerol utilization pathway components). Where two exogenous nucleicacids encoding glycerol utilization pathway components are introducedinto a host methanotrophic bacterium, it is understood that the twoexogenous nucleic acids can be introduced as a single nucleic acidmolecule, for example, on a single plasmid, on separate plasmids, can beintegrated into the host chromosome at a single site or multiple sites,and still be considered two exogenous nucleic acids. Similarly, it isunderstood that more than two exogenous nucleic acid molecules can beintroduced into a host bacterium in any desired combination, forexample, on a single plasmid, on separate plasmids, can be integratedinto the host chromosome at a single site or multiple sites, and stillbe considered as two or more exogenous nucleic acids. Thus, the numberof referenced exogenous nucleic acids or enzymatic activities refers tothe number of encoding nucleic acids or the number of proteinactivities, not the number of separate nucleic acid molecules introducedinto the host bacterium.

As used herein, “nucleic acid”, also known as polynucleotide, refers toa polymeric compound comprised of covalently linked subunits callednucleotides. Nucleic acids include polyribonucleic acid (RNA),polydeoxyribonucleic acid (DNA), both of which may be single or doublestranded. DNA includes cDNA, genomic DNA, synthetic DNA, andsemi-synthetic DNA.

As used herein, “multi-carbon substrate utilization pathway component”refers to any enzyme or protein that is involved (e.g., a protein orenzyme involved in transport or catalyzing an enzymatic reaction) in theability of an organism to utilize the selected multi-carbon substrate asa carbon and energy source. Exemplary multi-carbon substrate utilizationpathways include glucose, acetate, lactate, arabinose, citrate,succinate, and glycerol utilization pathways. Sources of nucleic acidsencoding multi-carbon substrate utilization pathway components are wellknown in the art and may be derived from a variety of species, includingbacteria, yeast, or other microorganisms.

As used herein, a “glucose transporter” refers to transport protein ortransport system that transports glucose into a microorganism. Exemplaryglucose transporters include phosphoenolpyruvate:sugarphosphotransferase system (PTS), which is involved in the uptake andconcomitant phosphorylation of a variety of hexose sugars in bacteria,and members of the major facilitator family (e.g., glucose/galactosetransporter (GluP), and galactose permease (GalP)).

As used herein, an “acetate transporter”, also known as “monocarboxylicacid transporter”, refers to a transport protein or transport systemthat transports acetate into a microorganism. Exemplary acetatetransporters include acetate permeases and proton-linked monocarboxylatetransporters of the sodium/solute symporter family (e.g., MctC, MctP,ActP).

As used herein, “acetyl-CoA synthase”, also known as “acetyl-CoAsynthetase”, refers to an enzyme that ligates acetate to coenzyme A toform acetyl coenzyme A (acetyl-CoA).

As used herein, a “lactate transporter”, also known as “monocarboxylicacid transporter”, refers to a transport protein or transport systemthat transports lactate into a microorganism. Exemplary lactatetransporters include lactate permease (e.g., LctP (also known as LacP)),and proton-linked monocarboxylate transporters of the sodium/solutesymporter family (e.g., MctC, MctP, ActP).

As used herein, a “lactate dehydrogenase”, refers to an NAD-independentenzyme that catalyzes the oxidation of lactate to pyruvate. As usedherein, lactate dehydrogenase includes L-lactate and D-lactatedehydrogenase.

As used herein, an “arabinose operon”, also known as “L-arabinoseoperon” or “ara operon”, refers to a gene sequence encoding enzymesneeded for the catabolism of arabinose to D-xylulose-5-phosphate, anintermediate of the pentose phosphate pathway.

As used herein, an “arabinose transporter” refers to a membranetransport protein or system that transports L-arabinose into amicroorganism. An exemplary arabinose transporter includes the lowaffinity AraE transport protein and the AraFGH ATP-binding cassette(ABC) transporter system. AraF is a periplasmic arabinose-bindingprotein, AraG is an ATP-binding component, and AraH is a membrane-boundcomponent.

As used herein, “glycerol utilization pathway component”, also known as“glycerol metabolism pathway enzyme” or “glycerol fermentation pathwayenzyme” refers to any enzyme or protein that is involved (e.g.,transport or catalyzing enzymatic reaction) in the ability of anorganism to utilize glycerol as a carbon and energy source. A glycerolutilization pathway component may be from an anaerobic pathway oraerobic pathway. A glycerol utilization pathway component includes, forexample, glycerol uptake facilitators, glycerol kinase,glycerol-3-phosphate dehydrogenase, glycerol dehydrogenase, ATP- orphosphoenolpyruvate-dependent dihydroxyacetone kinase. Sources ofnucleic acids encoding glycerol utilization pathway components are wellknown in the art and may be derived from a variety of species, includingbacteria, yeast, or other microorganisms.

As used herein, “glycerol uptake facilitator”, also known as “glycerolfacilitator” refers to a cytoplasmic membrane protein that transportsglycerol into a cell. It may be a member of the major intrinsic protein(MIP) family of transmembrane channel proteins. As used herein, aglycerol uptake facilitator may refer to a membrane protein thatfacilitates diffusion of or actively transports glycerol into a cell.The activity of glycerol uptake facilitator can be measured by atransport assay (see, e.g., Voegele and Boos, 1993, J. Bacteriol.175:1087-1094).

As used herein, “glycerol kinase”, also known as glycerokinase, refersto an enzyme that catalyzes the phosphorylation of glycerol toglycerol-3-phosphate (G3P). The activity of glycerol kinase can bemeasured as described in Lin et al., 1962, Proc. Natl. Acad. Sci. USA48:2145-2150.

As used herein, “glycerol-3-phosphate dehydrogenase” refers to an enzymethat catalyzes the oxidation of glycerol-3-phosphate (G3P) todihydroxyacetone phosphate. The activity of glycerol-3-phosphatedehydrogenase can be measured by the method of Spector and Pizer (1975,Methods Enzymol. 41:249-254). Glycerol-3-phosphate dehydrogenaseincludes both aerobic and anaerobic versions. In certain embodiments,the glycerol-3-phosphate is an aerobic enzyme.

As used herein, “transformation” refers to the transfer of a nucleicacid (e.g., exogenous nucleic acid) into the genome of a host bacterium,resulting in genetically stable inheritance. Host bacteria containingthe transformed nucleic acids are referred to as “recombinant” or“non-naturally occurring” or “genetically engineered” or “transformed”or “transgenic” bacteria.

Methanotrophic Bacteria

In certain embodiments, obligate methanotrophic bacteria are geneticallyengineered with the capability to utilize a multi-carbon substrate as acarbon and energy source. In other embodiments, facultativemethanotrophic bacteria are genetically engineered with the capacity toutilize a non-native multi-carbon substrate. Methanotrophic bacteriahave the ability to oxidize methane as a carbon and energy source.Methanotrophic bacteria are classified into three groups based on theircarbon assimilation pathways and internal membrane structure: type I(gamma proteobacteria), type II (alpha proteobacteria, and type X (gammaproteobacteria). Type I methanotrophs use the ribulose monophosphate(RuMP) pathway for carbon assimilation whereas type II methanotrophs usethe serine pathway. Type X methanotrophs use the RuMP pathway but alsoexpress low levels of enzymes of the serine pathway. Methanotrophicbacteria are grouped into several genera: Methylomonas, Methylobacter,Methylococcus, Methylocystis, Methylosinus, Methylomicrobium,Methanomonas, and Methylocella. Methanotrophic bacteria include obligatemethanotrophs, which can only utilize C1 substrates for carbon andenergy sources, and facultative methanotrophs, which naturally have theability to utilize some multi-carbon substrates as a sole carbon andenergy source. Facultative methanotrophs include some species ofMethylocella, Methylocystis, and Methylocapsa (e.g., Methylocellasilvestris, Methylocella palustris, Methylocella tundrae, Methylocystisdaltona strain SB2, Methylocystis bryophila, and Methylocapsa aureaKYG), and Methylobacterium organophilum (ATCC 27,886). Exemplaryobligate methanotrophic bacteria include: Methylococcus capsulatus Bathstrain, Methylomonas 16a (ATCC PTA 2402), Methylosinus trichosporiumOB3b (NRRL B-11,196), Methylosinus sporium (NRRL B-11,197),Methylocystis parvus (NRRL B-11,198), Methylomonas methanica (NRRLB-11,199), Methylomonas albus (NRRL B-11,200), Methylobacter capsulatus(NRRL B-11,201), Methylomonas flagellata sp AJ-3670 (FERM P-2400),Methylacidiphilum infernorum and Methylomicrobium alcaliphilum 20Z.

A selected methanotrophic host bacteria may also undergo strainadaptation under selective conditions to identify variants with improvedproperties for production. Improved properties may include increasedgrowth rate, yield of desired products, and tolerance of likely processcontaminants. In a particular embodiment, a high growth variantmethanotrophic bacteria, which is an organism capable of growth onmethane as the sole carbon and energy source and which possesses anexponential phase growth rate that is faster (i.e., shorter doublingtime) than its parent, reference, or wild-type bacteria, is selected(see, e.g., U.S. Pat. No. 6,689,601).

Each of the methanotrophic bacteria of this disclosure may be grown asan isolated pure culture, with a heterologous organism(s) that may aidwith growth, or one or more different strains of methanotrophic bacteriamay be combined to generate a mixed culture.

Multi-carbon Substrate Utilization Pathway Enzymes Glycerol UtilizationPathway Enzymes

Glycerol can be used as a source of carbon and energy by manymicroorganisms. As used herein, “glycerol utilization pathwaycomponent”, also known as “glycerol metabolism pathway enzyme” or“glycerol fermentation pathway enzyme” refers to any enzyme or proteinthat is involved in the ability of an organism to utilize glycerol as acarbon and energy source. A glycerol utilization pathway component mayinclude a number of enzymes and transport proteins from multipleglycerol utilization pathways, including for example, glycerol uptakefacilitators, glycerol kinase, glycerol-3-phosphate dehydrogenase,glycerol dehydrogenase, and dihydroxyacetone kinase.

The initial step of glycerol utilization is its uptake by themicroorganism. Glycerol can passively diffuse through membranes withouta transport system. However, many microorganisms possess specificglycerol transporters. Glycerol transport can be mediated by a glyceroluptake facilitator (facilitated diffusion), or by an active glyceroltransporter (e.g., ATP dependent transporter or proton glycerolsymporter). Genes that encode glycerol transporters include, forexample, glpF, glpT, aqp, gup1, gup2, mip, gtsA, gtsB, gtsC, and stl1.An exemplary amino acid sequence for a glycerol uptake facilitatorcomprises any one of SEQ ID NOS:1-21.

In most microorganisms that utilize glycerol, once glycerol has enteredthe cell, it is phosphorylated by glycerol kinase (GK) intoglycerol-3-phosphate (G3P), which is then oxidized byglycerol-3-phosphate dehydrogenase to form dihydroxyacetone phosphate(DHAP) (i.e., in presence of electron acceptors, respiratory metabolism)(see, FIG. 1). In the respiratory glycerol utilization pathway, thereare two forms of glycerol-3-phosphate dehydrogenase, aerobic andanaerobic (e.g., GlpD and GlpABC, respectively). Genes that encodeglycerol kinase include, for example, glpK, gut1, and gykA. An exemplaryamino acid sequence for a glycerol kinase comprises any one of SEQ IDNOS:22-42. Genes that encode glycerol-3-phosphate dehydrogenase include,for example, glpD, glpA, glpB, glpC, gpsA, glyC, and gpdA2. An exemplaryamino acid sequence for a glycerol-3-phosphate dehydrogenase comprisesany one of SEQ ID NOS:43-63. A small number of yeast and bacteria,however, use an alternative pathway in the absence of electron acceptors(i.e., fermentative metabolism), where glycerol is oxidized by glyceroldehydrogenase into dihydroxyacetone, which is then phosphorylated bydihydroxyacetone kinase into dihydroxyacetone phosphate.

Glucose Utilization Pathway Enzymes

Glucose utilization in bacteria is well characterized (see, e.g., Hughand Leifson, 1953, J. Bacteriol. 66:24-26; Tomlinson et al., 1974,Canadian J. Microbiol. 20:1085-1091; Takahashi and Yamada, 1999, Crit.Rev. Oral Biol. Med. 10:487-503; Fuhrer et al., 2005, J. Bacteriol.187:1581-1590; Goldman and Blumenthal, 1963, J. Bacteriol. 86:303-311;Fraenkel and Levisohn, 1967, J. Bacteriol. 93:1571-1578; Hua et al.,2003, J. Bacteriol. 185:7053-7067). The main pathway for glucosecatabolism is the Embden-Meyerhof-Parnas (EMP) pathway, a type ofglycolysis that converts glucose into pyruvate and is widely distributedin saccharolytic bacteria. Some bacteria may use another glycolyticpathway, the Entner-Doudoroff pathway, to convert glucose to pyruvate.An alternative pathway to glycolysis is the pentose phosphate pathwaywhich converts glucose to ribulose-5-phosphate in an oxidative phase. Anon-oxidative phase of the pentose phosphate pathway catalyzes theinterconversion of phosphorylated sugars to xylulose-5-phosphate,ribulose-5-phosphate, and ribose-5-phosphate. Glucose-6-phosphate is areaction component in the glycolytic pathways and pentose phosphatepathway.

Phosphoenolpyruvate:sugar phosphotransferase systems (PEP-sugar-PTS) aremulti-component systems involving enzymes of the plasma membrane and inthe cytoplasm that catalyze the concomitant transport andphosphorylation of hexose sugars (e.g., glucose) to hexose-6-phosphate(see, e.g., Herzberg and Klevit, 1994, Curr. Biol. 4:814-822; Takahashiand Yamada, 1999, Crit. Rev. Oral Biol. Med. 10:487-503; Saier, 1977,Bacteriological Rev. 41:856-871, disclosures of which are incorporatedherein by reference, in their entirety). Cytoplasmic proteins enzyme EIand Histidine Protein (HPr) initiate phosphoryl transfer reactions andfunction in the transport and phosphorylation of all sugar substrates ofthe system. Enzymes II are sugar-specific permeases or transporters,commonly consisting two cytoplasmic domains EIIA and EIIB and anintegral membrane domain EIIC. An exemplary amino acid sequence forcomponents of PEP-glucose-PTS systems from E. coli comprises any one ofSEQ ID NOS:64-67. Exemplary amino acid sequences for EI and HPr and EIIAcomponents are provided in SEQ ID NOs: 64 and 65. Exemplary EIIB andEIIC components are provided in SEQ ID NOs: 66 and 67.

Alternative mechanisms for glucose uptake include glucose ion symporters(see, e.g., Sarker et al., 1997, J. Bacteriol. 179:1805-1808; Essenberget al., 1997, Microbiology 143:1549-1555; Henderson et al., 1977,Biochem. J. 162:309-320, disclosures of which are incorporated herein byreference, in their entirety) and ABC transporters (see, e.g., Albers etal., 1999, J. Bacteriol. 14:4285-4291; Chevance et al., 2006, J.Bacteriol. 188:6561-6571; Wanner and Soppa, 1999, Genetics152:1417-1428, disclosures of which are incorporated herein byreference, in their entirety). An exemplary amino acid sequence for aglucose ion symporter or ABC transporter comprises any one of SEQ IDNOS:68-71. Glucose may be then be phosphorylated to glucose 6-phosphateby a separate gluco-kinase. An exemplary amino acid sequence for agluco-kinase comprises SEQ ID NO:72 or 73.

Acetate Utilization Pathway Enzymes

The utilization of acetate as a carbon and energy source has beenpreviously described (see, e.g., Gerstmeir et al., 2003, J. Biotechnol.104:99-122). To grow on acetate, bacteria activate it to acetyl-CoA.Acetate may be converted to acetyl-phosphate and then to acetyl-CoA viaacetate kinase and phosphotransacetylase enzymes, respectively. Acetatemay also be converted directly to acetyl-CoA by acetyl-CoA synthase. Anexemplary amino acid sequence for acetyl-CoA synthase comprises SEQ IDNO:74.

Bacterial transport systems for uptake of acetate include monocarboxylictransporters. Exemplary acetate transporters include ActP of E. coli(Gimenez et al., 2003, J. Bacteriol. 185:6448-6455, incorporated hereinby reference, in its entirety), MctP of Rhizobium leguminosarum (Hosieet al, 2002, J. Bacteriol. 184:5436-5448, incorporated herein byreference, in its entirety), and MctC of Corynebacterium glutamicum(Jokver et al., 2009, J. Bacteriol. 191:940-948, incorporated herein byreference, in its entirety). An exemplary amino acid sequence for anacetate transporter comprises SEQ ID NO:75.

Lactate Utilization Pathway Enzymes

Many bacteria are able to utilize D- or L-lactate as a sole source ofcarbon and energy (see, e.g., Chai et al., 2009, J. Bacteriol.191:2423-2430; Pinchuk et al., 2009, Proc. Natl. Acad. Sci. USA106:2874-2879; Bryant et al., 1977, Appl. Environ. Microbiol.33:1162-1169; Erwin and Gotschlich, 1993, J. Bacteriol. 175:6382-6391;Myers and Nealson, 1988, 240:1319-1321; Garvie, 1980, Microbiol. Rev.44:106-139). To use lactate as a source of carbon, it is oxidized topyruvate by lactate dehydrogenase. An exemplary amino acid sequence fora lactate dehydrogenase comprises SEQ ID NO:76.

Monocarboxylic transporters that transport acetate may also be capableof transporting lactate. Lactate transporters have been described andinclude, for example, LutP (formerly YvfH) (Chai et al., 2009, J.Bacteriol. 191:2423-2430, incorporated herein by reference, in itsentirety), MctP (Hosie et al., 2002, J. Bacteriol. 184:5436-5448,incorporated herein by reference, in its entirety), GlcA (YghK) and LctP(LldP) (Nunez et al, 2001, Microbiol. 147:1069-1077, incorporated hereinby reference, in its entirety). An exemplary amino acid sequence for alactate transporter comprises SEQ ID NO:77 or 78.

Arabinose Utilization Pathway Enzymes

The utilization of arabinose as a carbon and energy source has been wellcharacterized in a number of bacteria (see, e.g., Engelsberg et al.,1962, J. Bacteriol. 84:137-146; Brown et al., 1972, J. Bacteriol.111:606-613; Stoner et al., 1983, J. Mol. Biol. 170:1049-1053; Gallegoset al., 1997, Microbiol. Mol. Biol. Rev. 61:393-410; Schleif R., 2000,Trends Genetc. 16:559-565; Sa-Nogueira et al., 1997, Microbiol.143:957-969; Kawaguchi et al. 2009, Appl. Environ. Microbiol.75:3419-3429; Vlieg et al., 2006, Curr. Opin. Biotechnol. 17:183-190;U.S. Patent Publication 2011/0143408, disclosures of which areincorporated herein by reference, in their entirety). The arabinoseoperon, also known as the L-arabinose operon or ara operon, is a genesequence encoding enzymes needed for the catabolism of L-arabinose toD-xylulose 5-phosophate, an intermediate of the pentose phosphatepathway. The ara operon has both positive and negative regulation. In E.coli, the ara operon comprises of a regulator gene AraC, pC and pBADpromoters, and enzymes AraB, AraA, and AraD. AraA is an L-arabinoseisomerase that converts arabinose to L-ribulose. AraB is a kinase thatphosphorylates L-ribulose. AraD is an epimerase that convertsL-ribulose-5-phosphate to D-xylulose-5-phosphate. AraE is a low affinitytransporter that is bound to the inner membrane and uses theelectrochemical potential to transport arabinose. AraFGH genes encodearabinose-specific components of a high-affinity ABC transporter. AraFis the periplasmic arabinose-binding protein. AraG is the ATP-bindingcomponent, and AraH is the membrane bound component. AraC regulates thearabinose catabolic genes (AraBAD) through interactions with the pBADand pC promoter regions and is itself under arabinose-induced control(reviewed in Schleif R., 2002, SGM symposium 61: Signals, switches,regulons and cascades: control of bacterial gene expression. Ed. D. A.Hodgson, C. M. Thomas. Cambridge Univ. Press). Additional arabinosetransporters include an arabinose permease AraP and arabinosetransporter AraT. Exemplary amino acid sequences for AraB, AraA, AraD,AraE, AraF, AraG, and AraH comprise SEQ ID NOS: 79, 80, 81, 82, 83, 84,and 85, respectively.

Citrate Utilization Pathway Enzymes

Citrate metabolism pathways in bacteria have been well characterized(see, e.g., Martin et al., 2005, J. Bacteriol. 187:5146-5155; Drider etal., 2004, Genet. Mol. Res. 3:273-281; Bott, 1997, Arch. Microbiol.167:78-88; Bott et al., 1995, Mol Microbiol. 18:533-546; Yamamoto etal., 2000, Mol. Microbiol. 37:898-912; Korithoski et al., 2005, J.Bacteriol. 2005, 187:4451-4456; Vlieg et al., 2006, Curr. Opin.Biotechnol. 17:183-190). Citrate uptake is mediated by citratetransporters including, for example, CitM (Warner et al., 2000, J.Bacteriol. 182:6099-6105; Korithoski et al., 2005, J. Bacteriol.187:4451-4456, disclosures of which are incorporated herein byreference, in their entirety), CitS (van der Rest et al., 1992, J. Biol.Chem. 267:8971-8976; Lolkema et al., 1994, Eur. J. Biochem. 220:469-475,disclosures of which are incorporated herein by reference, in theirentirety), CitP (Magni et al., 1996, FEMS Microbiol. Lett. 142:265-269,incorporated herein by reference, in its entirety), CitC (Ishiguro etal., 1992, J. Bio. Chem. 267:9559-9564, incorporated herein byreference, in its entirety), and CitH (Lolkema et al., 1994, Eur. J.Biochem. 220:469-475, incorporated herein by reference, in itsentirety). An exemplary amino acid sequence for a citrate transportercomprises SEQ ID NO:86.

Succinate Utilization Pathway Enzymes

Bacterial utilization of succinate as a carbon and energy source hasbeen described for a variety of bacteria (see, e.g., Janssen andLiesack, 1995, Arch. Microbiol. 164:29-35; Denger and Schink, 1990,Arch. Microbiol. 154:550-555; Jansen, 1991, Arch. Microbiol.155:288-293; Gylswiyk et al., 1997, Int. J. Syst. Bacteriol. 47:155-9;Duetz et al., 1994, J. Bacteriol. 176:2354-2361).

Succinate is a component of the citric acid cycle or glyoxylate cyclefor generating energy. Succinate is oxidized by succinate dehydrogenaseto fumarate. In some bacteria, succinate is decarboxylated to propionateand CO₂ by methylmalonyl-CoA decarboxylase (see, e.g., Bott et al.,1997, Eur. J. Biochem. 250:590-599; Ruiz-Herrera and Garcia, 1972, J.Gen. Microbiol. 72:29-35).

Transport systems for C₄-dicarboxylates (e.g., succinate) have beendescribed in a number of bacteria. For example, E. coli use anaerobicDcuA and DcuB and aerobic Dct dicarboxylate transport systems (Lo etal., 1977, J. Supramol. Struct. 7:463-480; Six et al., 1994, J.Bacteriol. 176:6470-6478, disclosures of which are incorporated hereinby reference, in their entirety). YdbFG sensor-regulator and YdbHC₄-dicarboxylate transport protein have been described in Bacillussubtilis (Asai et al., 2000, Microbiol. 146:263-271, incorporated hereinby reference, in its entirety). Rhodobacter capsulatus has a Dcttransport system which consists of three proteins: C₄-dicarboxylateperiplasmic binding protein, DctP, and two integral membrane proteins,DctQ and DctM (Forward et al., 1994, J. Bacteriol. 179:5482-5493,incorporated herein by reference, in its entirety). DcsT mediates uptakeof C4 dicarobxylates, including succinate, in Corynebacterium glutamicum(Teramoto et al., 2008, Appl. Environ. Microbiol. 74:5290-5296,incorporated herein by reference, in its entirety). An exemplary aminoacid sequence for a succinate transporter comprises SEQ ID NO:87 or 88.

Recombinant Methanotrophic Bacteria

Provided in the present disclosure are recombinant methanotrophicbacteria that may be produced by introducing (e.g., by transformation)into the host bacteria at least one expressible exogenous nucleic acidencoding a multi-carbon substrate utilization pathway component.Alternatively, if a selected methanotrophic host bacterium exhibitsendogenous expression of one of more genes of a multi-carbon substrateutilization pathway, but is deficient in others, then an encodingnucleic acid is needed for the deficient component(s) to achieve thedesired multi-carbon substrate utilization capability. Thus, arecombinant methanotrophic bacterium of the invention can be produced byintroducing exogenous component activities to obtain a desiredmulti-carbon substrate utilization pathway or a desired multi-carbonsubstrate utilization pathway can be obtained by introducing one or moreexogenous component activities which, together with one or moreendogenous components, allow use of a multi-carbon substrate as a carbonsource. However, it is understood that even if a host methanotrophicbacterium contains at least one multi-carbon substrate utilizationpathway component, introduction of exogenous nucleic acids encodingcomponents of a complete multi-carbon substrate utilization pathway maybe included. In some embodiments, a recombinant methanotrophic bacteriumas described herein can also include other genetic modifications thatfacilitate or optimize a multi-carbon substrate utilization pathway orthat confer other useful functions onto the host. For example, if aselected host methanotrophic bacteria exhibits endogenous expression ofa protein or enzyme that inhibits or competes with a multi-carbonsubstrate utilization pathway, then the host may be genetically modifiedso that it does not produce a functional protein or enzyme or asubstantial amount of a functional protein or enzyme that inhibits orcompetes with the desired multi-carbon substrate utilization. In anotherexample, selected host methanotrophic bacteria may be geneticallymodified to increase expression of an endogenous gene that enhancesutilization of a desired multi-carbon substrate. Additionally, a hostmethanotrophic bacterium may possess other genetic modificationsconferring it with other desirable properties, unrelated to multi-carbonsubstrate utilization. For example, a host methanotrophic bacterium maypossess genetic modifications conferring high growth, tolerance ofcontaminants or particular culture conditions, ability to metabolizeadditional carbon substrates, or ability to synthesize desirableproducts or intermediates (e.g., propylene, crotonate, or crotonyl CoA,see, e.g., International Application Number PCT/US13/60460, incorporatedherein by reference, in its entirety).

In certain embodiments, the present disclosure provides recombinantobligate methanotrophic bacteria or recombinant facultativemethanotrophic bacteria including at least one exogenous nucleic acidencoding a multi-carbon substrate utilization pathway component, whereinthe multi-carbon substrate is not utilized as a carbon source by areference facultative methanotrophic bacterium, wherein the at least oneexogenous nucleic acid encoding a multi-carbon substrate utilizationpathway component is expressed in a sufficient amount to permit growthof the recombinant obligate methanotrophic bacteria on the multi-carbonsubstrate as a primary carbon source or the recombinant facultativemethanotrophic bacteria on the multicarbon substrate as a sole carbonsource. A multi-carbon substrate may be a carbohydrate or organic acid,including, for example, glucose, acetate, lactate, arabinose, citrate,succinate, and glycerol. Recombinant obligate methanotrophic bacteriause a selected multi-carbon substrate as a primary carbon source if theselected multi-carbon substrate is the source of at least 50% or more ofcarbon usage for the bacteria. In certain embodiments, the at least oneexogenous nucleic acid encoding a multi-carbon substrate utilizationpathway component is expressed in a sufficient amount to permit growthof the recombinant obligate methanotrophic bacteria on the multi-carbonsubstrate as a sole carbon source.

A reference facultative methanotrophic bacterium, also known as parentor wildtype facultative methanotrophic bacterium, is one that has notbeen genetically engineered with the capability to use an additionalmulti-carbon substrate other than its native substrates. A referencefacultative methanotrophic bacterium may be selected for geneticengineering to introduce at least one exogenous nucleic acid encoding amulti-carbon substrate utilization pathway component for a multi-carbonsubstrate it does not naturally utilize (i.e., cannot grow on theselected multi-carbon substrate as sole carbon source) and becomes arecombinant facultative methanotrophic bacterium.

In certain embodiments wherein the multi-carbon substrate is glucose,recombinant methanotrophic bacteria include an exogenous nucleic acidencoding a glucose transporter. A number of methanotrophic bacterialgenomes encode the enzymes of a complete glycolysis pathway necessaryfor utilization of D-glucose (see, e.g., Stein et al., 2010, J.Bacteriol. 192:6497-6498; U.S. Pat. No. 6,555,353; Ward et al., 2004,PLoS Biol. 2:e303; Vuilleumier et al., 2012, J. Bacteriol. 194:551-552).Methanotrophs have also been found to possess pentose phosphate pathwaygenes (Dedysh et al., 2000, Int. J. Syst. Evol. Microbiol. 50:955-969;Vuilleumier et al., 2012, J. Bacteriol. 194:551-552). However,methanotrophic bacteria lack glucose transporters that allow them tobring extracellular sugars into the cell, as well as enzymes tophosphorylate glucose. A glucose transporter suitable for introductioninto a methanotrophic bacteria may be a phosphoenolpyruvate:glucosephosphotransferase system, a glucose ion symporter, or an ABCtransporter. Exemplary amino acid sequences forphosphoenolpyruvate:glucose phosphotransferase system componentscomprise a sequence provided by SEQ ID NOS:64-67. Exemplary EI, HPr, andEIIA components comprise amino acid sequences provided by SEQ ID NO: 64or 65; exemplary EIIC and EIIB components comprise amino acid sequencesprovided by SEQ ID NOs: 66 or 67. An exemplary amino acid sequence forglucose ion symporters and ABC transporters comprises SEQ ID NO:68, 69,70, or 71. If the glucose transporter is not aphosphoenolpyruvate:glucose phosphotransferase system, then therecombinant methanotrophic bacterium may further include an exogenousnucleic acid encoding a gluco-kinase to phosphorylate imported glucose.An exemplary amino acid sequence for gluco-kinase comprises SEQ ID NO:72or 73.

In certain embodiments wherein the multi-carbon substrate is acetate,recombinant methanotrophic bacteria include an exogenous nucleic acidencoding an acetate transporter. Examples of acetate transportersinclude ActP, MctP, and MctC. An exemplary amino acid sequence for anacetate transporter comprises SEQ ID NO:75. Methanotrophic bacteria lackacetate transporters but possess an acetyl-CoA synthase gene, whichactivates acetate to acetyl-CoA. Metabolism of acetate may be feasibleif it is transported into the cell. In certain embodiments, therecombinant methanotrophic bacteria comprising an exogenous nucleic acidencoding an acetate transporter is further modified to overexpressacetyl-CoA synthase. Up-regulation or overexpression of an endogenous orexogenous nucleic acid encoding acetyl-CoA synthase may improvemethanotrophic bacterial growth rate on exogenous acetate, as theendogenous enzyme may not be expressed at optimal levels for growth onthis non-native substrate. An exemplary amino acid sequence for anacetyl-CoA synthase comprises SEQ ID NO:74.

In certain embodiments, wherein the multi-carbon substrate is lactate,recombinant methanotrophic bacteria include an exogenous nucleic acidencoding a lactate transporter and an exogenous nucleic acid encoding alactate dehydrogenase. Examples of lactate transporters include LutP(formerly YvfH), MctP, GlcA (YghK), and LctP (LldP). A number ofmonocarboxylic acid transporters that transport acetate are capable oftransporting lactate also. An exemplary amino acid sequence for lactatetransporter comprises SEQ ID NO:77 or 78. A nucleic acid encodinglactate dehydrogenase is introduced into recombinant methanotrophicbacteria to convert imported lactate to pyruvate, which may then enterendogenous pyruvate metabolic pathways of the recombinant methanotrophicbacteria. An exemplary amino acid sequence for lactate dehydrogenasecomprises SEQ ID NO:76.

In certain embodiments, wherein the multi-carbon substrate is arabinose,recombinant methanotrophic bacteria include an exogenous nucleic acidencoding an L-arabinose isomerase (e.g., AraA), an exogenous nucleicacid encoding an L-ribulose kinase (e.g., AraB), an exogenous nucleicacid encoding a L-ribulose-5-phosphate epimerase (e.g., AraD), and anexogenous nucleic acid encoding an arabinose transporter. Exemplaryamino acid sequences for AraB, AraA, and AraD comprise SEQ ID NOs: 79,80, and 81, respectively. An arabinose transporter, for example, AraE,AraFGH, or AraP, is used to transport arabinose into the recombinantmethanotrophic bacteria. Exemplary AraE, AraF, and AraG, and AraH aminoacid sequences comprise SEQ ID NOs: 82, 83, 84, and 85, respectively.AraA is an L-arabinose isomerase that converts arabinose to L-ribulose.AraB is a kinase that phosphorylates L-ribulose. AraD is an epimerasethat converts L-ribulose-5-phosphate to D-xylulose-5-phosphate.D-xylulose-5-phosphate is a pre-cursor to ribulose-5-phosphate, a keyintermediate in the ribulose monophosphate (RuMP) pathway employed byType I and Type X methanotrophic bacteria, and is likely to be usedefficiently by recombinant methanotrophic bacteria. In furtherembodiments, the L-arabinose isomerase is AraA, the L-ribulose kinase isAraB, and L-ribulose-5-phosphate epimerase is AraD, and the arabinosetransporter is AraE, AraFGH, or AraP.

In certain embodiments, wherein the multi-carbon substrate is citrate,recombinant methanotrophic bacteria include an exogenous nucleic acidencoding a citrate transporter. Examples of citrate transporters includeCitM, CitS, CitP, CitC, and CitH. An exemplary amino acid sequence for acitrate transporter comprises SEQ ID NO:86. Once citrate is importedinto the cell, recombinant methanotrophs are expected to grow, as theypossess enzymes for utilizing citrate.

In certain embodiments, wherein the multi-carbon substrate is succinate,recombinant methanotrophic bacteria include an exogenous nucleic acidencoding a succinate transporter. Examples of succinate transportersinclude DcuA, DcuB, Dct, YdbH, Dct, and DcsT. An exemplary amino acidsequence for a succinate transporter comprises SEQ ID NO:87 or 88. Oncesuccinate is imported into the cell, recombinant methanotrophs areexpected to grow, as they possess enzymes for succinate utilization.

In certain embodiments wherein the multi-carbon substrate is glycerol,the recombinant methanotrophic bacteria includes at least two exogenousnucleic acids encoding glycerol utilization pathway components. Incertain embodiments the at least two exogenous nucleic acids areexpressed in an amount sufficient to permit growth of the recombinantmethanotrophic bacteria on glycerol as a sole carbon source. In certainembodiments, the at least two glycerol utilization pathway componentscomprise components that are from the respiratory glycerol metabolismpathway. In certain embodiments, the at least two glycerol utilizationpathway components comprise glycerol kinase and glycerol-3-phosphatedehydrogenase. The glycerol-3-phophate dehydrogenase may be preferably,aerobic, or anaerobic. In a specific embodiment, the glycerol kinase isGlpK and the glycerol-3-phosphate dehydrogenase is GlpD. Exemplaryglycerol kinase and glycerol-3-phosphate dehydrogenase amino acidsequences encoded by exogenous nucleic acids that may be used totransform host methanotrophic bacteria comprise any one of SEQ IDNOS:22-42 and SEQ ID NOS:43-63, respectively. In certain embodiments,the glycerol kinase comprises an amino acid sequence of SEQ ID NO:22 andthe glycerol-3-phosphate dehydrogenase comprises an amino acid sequenceof SEQ ID NO:43.

In certain embodiments, recombinant methanotrophic bacteria comprisethree exogenous nucleic acids encoding glycerol utilization pathwaycomponents. The three exogenous nucleic acids encoding glycerolutilization pathway components may be expressed in an amount sufficientto permit growth of the recombinant methanotrophic bacterium on glycerolas a primary carbon source or as a sole carbon source. In certainembodiments, the three exogenous nucleic acids encoding glycerolutilization pathway components comprise glycerol uptake facilitator,glycerol kinase, and glycerol-3-phosphate dehydrogenase. In a specificembodiment, the glycerol uptake facilitator is GlpF, the glycerol kinaseis GlpK, and the glycerol-3-phosphate dehydrogenase is GlpD. Exemplaryglycerol uptake facilitator, glycerol kinase, and glycerol-3-phosphatedehydrogenase amino acid sequences encoded by exogenous nucleic acidsthat may be used to transform host methanotrophic bacteria comprise asequence selected from SEQ ID NOS:1-21, SEQ ID NOS:22-42, and SEQ IDNOS:43-63, respectively. In certain embodiments, the glycerol uptakefacilitator comprises an amino acid sequence of SEQ ID NO:1, theglycerol kinase comprises an amino acid sequence of SEQ ID NO:22, andthe glycerol-3-phosphate dehydrogenase comprises an amino acid sequenceof SEQ ID NO:43.

Recombinant methanotrophic bacteria comprising at least two exogenousnucleic acids encoding glycerol utilization pathway components, asdescribed herein, are expected to exhibit rapid and efficient growth inthe presence of glycerol. However, in the absence of glycerol (e.g.,during growth on methane as a sole carbon source), it is possible for aglycerol utilization pathway to run in reverse, where intracellular DHAPfrom gluconeogenesis is reduced to glycerol-3-phosphate byglycerol-3-phosphate dehydrogenase, which is capable of catalyzing areversible redox reaction. Glycerol-3-phosphate may then bede-phosphorylated by glycerol-3-phosphatase into glycerol. Glycerol maythen be secreted from the cell, thereby lowering the cellular growthrate. Therefore, in certain embodiments, expression of nucleic acidsencoding glycerol utilization pathway components may be regulated (e.g.,via inducible or repressible promoter) to provide for optimal bacterialgrowth under a variety conditions (e.g., presence of a particular carbonsource). For example, expression of nucleic acids encoding glycerolutilization pathway components may be regulated so that they are notexpressed in the absence of glycerol and expressed in the presence ofglycerol.

In certain embodiments, recombinant methanotrophic bacteria of any ofthe embodiments disclosed herein comprise two, three, four, five, six,or more exogenous nucleic acids encoding multi-carbon substrateutilization pathway components, wherein the exogenous nucleic acids areexpressed in an amount sufficient to permit growth of the recombinantmethanotrophic bacteria on the multi-carbon substrate as a primary orsole carbon source. Each exogenous nucleic acid may encode a differenttype of multi-carbon substrate utilization pathway component (i.e.,catalyze different enzymatic reactions or processes), or one or morenucleic acids may encode the same type of multi-carbon substrateutilization pathway component. For example, a recombinant methanotrophicbacterium may comprise two or more exogenous nucleic acids encoding aglycerol uptake facilitator, with each having a different sequence, inorder to increase glycerol import capability of the bacterium. It isapparent to one of skill in the art that any combination of two or morenucleic acids encoding multi-carbon substrate utilization pathwaycomponents may be used to constitute a multi-carbon substrateutilization pathway in a recombinant methanotrophic bacterium, providedthat the genetically engineered metabolic pathway provides therecombinant methanotrophic bacterium with the capability to metabolizethe selected multi-carbon substrate (e.g., glycerol intodihydroxyacetone phosphate, arabinose to D-xylulose 5-phosophate).

In certain embodiments, the recombinant methanotrophic bacteriaaccording to any of the embodiments disclosed herein is Methylomonas,Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, orMethanomonas. In further embodiments, the recombinant methanotrophicbacteria is Methylosinus trichosporium strain OB3b, Methylococcuscapsulatus Bath strain, Methylomonas methanica 16A strain, Methylosinustrichosporium (NRRL B-11,196), Methylosinus sporium (NRRL B-11,197),Methylocystis parvus (NRRL B-11,198), Methylomonas methanica (NRRLB-11,199), Methylomonas albus (NRRL B-11,200), Methylobacter capsulatus(NRRL B-11,201), Methylomonas sp AJ-3670 (FERM P-2400),Methylacidiphilum infernorum, or Methylomicrobium alcaliphilum 20Z.

In certain embodiments, the recombinant methanotrophic bacteriaaccording to any of the embodiments disclosed herein is Methylocellasilvestris, Methylocella palustris, Methylocella tundra, Methylocystisdaltona, Methylocystis bryophila, Methylobacterium organophilum (ATCC27,886), or Methylocapsa aurea.

Sources of encoding nucleic acids for multi-carbon substrate utilizationpathway components may include any bacterial, yeast, or othermicroorganism species where the encoded gene product is capable ofcatalyzing the referenced reaction in the multi-carbon substrateutilization pathway. Exemplary species for such sources are well knownin the art.

Exemplary sources of encoding nucleic acids for glucose utilizationpathway components include: Escherichia coli, Bacillus subtilis,Corynebacterium glutamicum, Saccharomyces cerevisiae, Zymomonas mobilis;Agrobacterium tumefaciens, Sinorhizobium meliloti; Rhodobactersphaeroides; Paracoccus versutus; Pseudomonas fluorescens, Pseudomonasputida, Salmonella enterica, Escherichia fergusonii, Salmonella enteric,Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica,Shigella flexneri, Shigella sonnei, Shigella boydii, Shigelladysenteriae, Pectobacterium atrosepticum, Pectobacterium wasabiae,Erwinia tasmaniensis, Erwinia pyrifoliae, Erwinia amylovora, Erwiniabillingiae, Buchnera aphidicola, Enterobacter sp. 638, Enterobactercloacae, Enterobacter asburiae, Enterobacter aerogenes, Cronobactersakazakii, Cronobacter turicensis, Klebsiella pneumoniae, Klebsiellavariicola, Klebsiella oxytoca, Citrobacter koseri, Citrobacterrodentium, Serratia proteamaculans, Serratia sp. AS12, Proteusmirabilis, Edwardsiella ictaluri, Edwardsiella tarda, CandidatusHamiltonella defense, Dickeya dadantii, Dickeya zeae, Pantoea anantis,Pantoea sp. At-9b, Pantoea vagans, Rahnella sp. Y9602, Haemophilusparasuis, Haemophilus parainfluenzae, Pasteurella multocida,Aggregatibacter aphrophilus, Aggregatibacter actinomycetemcomitans,Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrioharveyi, Vibrio splendidus, Photobacterium profundum, Vibrioanguillarum, Shewanella oneidensis, Shewanella denitrificans, Shewanellafrigidimarina, Shewanella amazonensis, Shewanella baltica, Shewanellaloihica, Shewanella sp. ANA-3, Shewanella sp. MR-7, Shewanellaputrefaciens, Shewanella sediminis, Shewanella sp. MR-4, Shewanella sp.W3-18-1, Shewanella woodyi, Psychromonas ingrahamii, Ferrimonasbalearica, Aeromonas hydrophile, Aeromonas salmonicida, Aeromonasveronii, Tolumonas auensis, Chromobacterium violaceum, Burkholderia sp.CCGE1002, Azospirillum sp. B510, Bacillus anthracis, Bacillus cereus,Bacillus cytotoxicus, Bacillus thuringiensis, Bacillusweihenstephanensis, Bacillus pseudofirmus, Bacillus megaterium,Staphylococcus aureus, Exiguobacterium sibiricum, Exiguobacterium sp.AT1b, Macrococcus caseolyticus, Paenibacillus polymyxa, Streptococcuspyogenes, Streptococcus pneumoniae, Streptococcus agalactiae,Streptococcus mutans, Streptococcus thermophilus, Streptococcussanguinis, Streptococcus suis, Streptococcus gordonii, Streptococcusequi, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcusgallolyticus, Streptococcus mitis, Streptococcus pseudopneumoniae,Lactobacillus johnsonii, Lactobacillus gasseri, Enterococcus faecalis,Aerococcus urinae, Carnobacterium sp. 17-4, Clostridium acetobutylicum,Clostridium perfringens, Clostridium tetani, Clostridium novyi,Clostridium botulinum, Desulfotomaculum reducens, Clostridiumlentocellum, Erysipelothrix rhusiopathiae, Mycoplasma genitalium,Mycoplasma pneumoniae, Mycoplasma pulmonis, Mycoplasma penetrans,Mycoplasma gallisepticum, Mycoplasma mycoides, Mycoplasma synoviae,Mycoplasma capricolum, Mycoplasma crocodyli, Mycoplasma leachii,Mesoplasma forum, Propionibacterium acnes, Nakamurella multipartita,Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii.

Exemplary sources of encoding nucleic acids for acetate utilizationpathway components include: Corynebacterium glutamicum, Escherichiacoli, and Rhizobium leguminosarum.

Exemplary sources of encoding nucleic acids for lactate utilizationpathway components include: Rhizobium leguminosarum, Bacillus subtilis,Staphylococcus aureus, Escherichia coli, Enterobacteriaceae,Propionibacterium pentosaceum, Pseudomonas aeruginosa, Acetobacterperoxydans, Selenomonas ruminantium, Pseudomonas natriegens, Aerobacteraerogenes, Lactobacillus casei, Lactobacillus plantarum, Serratia,Aerobacter cloacae, Proteus vulgaris, Escherichia freundii, Klebsiellasp., Hafnia sp., Butyribacterium rettgeri, Streptococcus faecium,Streptococcus lactis, Pediococcus pentosaceum, Salmonella typhimurium,Aggregatibacter actinomycetemcomitans, and Neisseria gonorrhoeae.

Sources of encoding nucleic acids for arabinose utilization pathwaycomponents include: Escherichia coli, Bacillus subtilis, Scheffersomycesstipitis, Corynebacterium glutamicum, Lactococcus lactis, Pichiastipitis, Shigella flexneri, Shigella boydii, Shigella dysenteriae,Salmonella typhimurium, Salmonella enterica, Klebsiella pneumoniae,Klebsiella oxytoca, Enterobacter cancerogenus, Bacillusamyloliquefaciens, Rhizobium, and Agrobacterium.

Sources of encoding nucleic acids for citrate utilization pathwaycomponents include: Lactococcus lactis, Enterococcus, Lactobacillusplantarum, Oenococcus oeni, Leuconostoc mesenteroides, Weissella,Salmonella dublin, Salmonella pulloru, Salmonella enteritidis,Klebsiella pneumoniae, Salmonella typhimurium, and Bacillus subtilis.

Sources of encoding nucleic acids for succinate utilization pathwaycomponents include: Escherichia coli, Corynebacterium glutamicum,Rhodobacter capsulatus, and Bacillus subtilis.

Sources of encoding nucleic acids for glycerol utilization pathwaycomponents include, for example: Escherichia coli, Acinetobacterbaumannii, Fusobacterium nucleatum subsp. vincentii, Pantoea sp. Scl,Pseudomonas aeruginosa, Shigella flexneri, Shewanella baltica OS155,Actinobacillus pleuropneumoniae serovar 3 str. JL03, Salmonella entericasubsp. enterica serovar Saintpaul str. SARA29, Yersinia bercovieri,Aeromonas veronii B565, Pseudomonas fluorescens, Serratia sp. AS12,Vibrio fischeri SR5, Haemophilus haemolyticus, Vibrio harveyi, Vibriocholera, Pseudomonas putida S16, Pectobacterium carotovorum subsp.carotovorum PC1, Pseudomonas syringae, Acinetobacter sp. ATCC 27244,Photobacterium profundum SS9, Citrobacter freundii, Klebsiellapneumoniae, Enterobacter sp., Enterococcus casseliflavus, Enterococcusfaecalis, Bacillus stearothermophilus, Bacillus subtilis, Streptococcuspyogenes, Haemophilus, influezae, Mycoplasma genitalium, Mycoplasmapneumonia, Mycoplasma mycoides, Yersinia mollaretii, Shigelladysenteriae, Shigella boydii, Shigella sonnei, Yersinia pestis, Yersiniaintermedia, Yersinia frederiksenii, Serratia proteamaculans, Erwiniacarotovora, Pseudomonas tolaasii, Yersinia enterolitica, Photorhabdusluminesens, Azotobacter vinelandii, Haemophilus ducreyi, Actinobacilluspleuropneumoniae, Aeromonas hydrophile, Photobacterium profundum,Aeromonas salmonicida, Vibrio angustum, Vibrio vulnificus, Vibrionalesbacterium, Vibrio splendidus, Vibrio sp. Ex25, Vibrio alginolyticus,Vibrio parahaemolyticus, Shewanella sp. W3-18-1, Alteromonas macleodii,Sodalis glossinidius, Pasteurella multocida, Salmonella typhimurium,Lactobacillus casei, Rhadopseudomonas, Propionibacterium, Nocardiaasteroides, Klebsiella aerogenes, Halobacterium cutirubrum,Gluconobacter oxydans, Staphylococcus aureus, Candida utilis, Candiamycodema, Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyceslactis, Ashbya gossypii, Lodderomyces elongisporus, Debaryomyceshansenii, Candida albicans, Pichia guilliermondii, Pichia stipitis, andFusarium oxysporum.

However, with the complete genome sequence available for hundreds ofmicroorganisms, the identification of genes encoding the requisitemulti-carbon substrate utilization pathway in related or distantspecies, including for example, homologs, orthologs, paralogs, etc., isroutine and well known in the art. Accordingly, exogenous nucleic acidsencoding multi-carbon substrate utilization pathway components describedherein with reference to particular nucleic acids from a particularorganism can readily include other nucleic acids encoding multi-carbonsubstrate utilization pathway components from other microorganisms. Forrecombinant methanotrophic bacteria comprising at least two exogenousnucleic acids encoding multi-carbon substrate utilization components,each nucleic acid may be derived from the same microorganism or fromdifferent microorganisms.

Polypeptide sequences and encoding nucleic acids for proteins, proteindomains, and fragments thereof described herein, such as a component ofa multi-carbon substrate utilization pathway, may also include naturaland recombinantly engineered variants. A nucleic acid variant refers toa nucleic acid that may contain one or more substitutions, additions,deletions, insertions, or may be or comprise fragment(s) of a referencenucleic acid. A reference nucleic acid refers to a selected nucleic acidencoding a multi-carbon substrate utilization pathway component. Avariant nucleic acid may have 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%,72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toa reference nucleic acid, as long as the variant nucleic acid can stillperform its requisite function or biological activity in themulti-carbon substrate utilization pathway (e.g., membrane transport). Avariant polypeptide may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity to a reference protein, as longas the variant polypeptide can still perform its requisite function orbiological activity (e.g., membrane transport). In certain embodiments,a multi-carbon substrate utilization pathway component that isintroduced into recombinant methanotrophs as provided herein encodes anamino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selectedfrom SEQ ID NOS:1-88. In certain embodiments, an exogenous nucleic acidencoding a multi-carbon substrate utilization pathway component that isintroduced into recombinant methanotrophs comprises a nucleic acidsequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identity to a nucleic acid sequence provided in Tables14-22. These variants may have improved function and biological activity(e.g., higher enzymatic activity or improved specificity for substrate)than the parent (or wildtype) protein. Due to redundancy in the geneticcode, nucleic acid variants may or may not affect amino acid sequence. Anucleic acid variant may also encode an amino acid sequence comprisingone or more conservative substitutions compared to a reference aminoacid sequence. A conservative substitution may occur naturally in thepolypeptide (e.g., naturally occurring genetic variants) or may beintroduced when the polypeptide is recombinantly produced. Aconservative substitution is where one amino acid is substituted foranother amino acid that has similar properties, such that one skilled inthe art would expect that the secondary structure and hydropathic natureof the polypeptide to be substantially unchanged. Amino acidsubstitutions may generally be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, and/or the amphipathicnature of the residues, and is known in the art. Amino acidsubstitutions, deletions, and additions may be introduced into apolypeptide using well-known and routinely practiced mutagenesis methods(see, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 3ded., Cold Spring Harbor Laboratory Press, NY 2001).Oligonucleotide-directed site-specific (or segment specific) mutagenesisprocedures may be employed to provide an altered polynucleotide that hasparticular codons altered according to the substitution, deletion, orinsertion desired. Deletion or truncation variants of proteins may alsobe constructed by using convenient restriction endonuclease sitesadjacent to the desired deletion. Alternatively, random mutagenesistechniques, such as alanine scanning mutagenesis, error prone polymerasechain reaction mutagenesis, and oligonucleotide-directed mutagenesis maybe used to prepare polypeptide variants (see, e.g., Sambrook et al.,supra). Variant nucleic acids may be naturally occurring or geneticallyengineered.

Nucleic acids encoding multi-carbon substrate utilization pathwaycomponents may be combined with other nucleic acid sequences, such aspromoters, polyadenylation signals, restriction enzyme sites, multiplecloning sites, other coding segments, and the like.

Differences between a wild type (or parent or reference) nucleic acid orpolypeptide and the variant thereof, may be determined by methodsroutinely practiced in the art to determine identity, which are designedto give the greatest match between the sequences tested. Methods todetermine sequence identity can be applied from publicly availablecomputer programs. Computer program methods to determine identitybetween two sequences include, for example, BLASTP, BLASTN (Altschul, S.F. et al., J. Mol. Biol. 215: 403-410 (1990), and FASTA (Pearson andLipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988). The BLAST familyof programs is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.

Assays for determining whether a polypeptide variant folds into aconformation comparable to the non-variant polypeptide or fragmentinclude, for example, the ability of the protein to react with mono- orpolyclonal antibodies that are specific for native or unfolded epitopes,the retention of ligand-binding functions, the retention of enzymaticactivity (if applicable), and the sensitivity or resistance of themutant protein to digestion with proteases (see Sambrook et al., supra).Polypeptides, variants and fragments thereof, can be prepared withoutaltering a biological activity of the resulting protein molecule (i.e.,without altering one or more functional activities in a statisticallysignificant or biologically significant manner). For example, suchsubstitutions are generally made by interchanging an amino acid withanother amino acid that is included within the same group, such as thegroup of polar residues, charged residues, hydrophobic residues, and/orsmall residues, and the like. The effect of any amino acid substitutionmay be determined empirically merely by testing the resulting modifiedprotein for the ability to function in a biological assay, or to bind toa cognate ligand or target molecule.

Codon Optimization

Expression of recombinant proteins is often difficult outside theiroriginal host. For example, variation in codon usage bias has beenobserved across different species of bacteria (Sharp et al., 2005, Nucl.Acids. Res. 33:1141-1153). Over-expression of recombinant proteins evenwithin their native host may also be difficult. In certain embodiments,at least one nucleic acid encoding a multi-carbon substrate utilizationpathway component that is to be introduced into host methanotrophicbacteria according to any of the embodiments described herein is codonoptimized to enhance protein expression in the methanotrophic bacteria.Codon optimization refers to alteration of codons in genes or codingregions of nucleic acids for transformation of a methanotrophicbacterium to reflect the typical codon usage of the host bacteriaspecies without altering the polypeptide for which the DNA encodes.Codon optimization methods for optimum gene expression in heterologoushosts have been previously described (see, e.g., Welch et al., 2009,PLoS One 4:e7002; Gustafsson et al., 2004, Trends Biotechnol.22:346-353; Wu et al., 2007, Nucl. Acids Res. 35:D76-79; Villalobos etal., 2006, BMC Bioinformatics 7:285; U.S. Patent Publication2011/0111413; U.S. Patent Publication 2008/0292918; disclosure of whichare incorporated herein by reference, in their entirety). One, two,three, or more nucleic acids encoding multi-carbon substrate utilizationpathway components may be codon optimized. For example, wherein arecombinant methanotrophic bacterium comprises two glycerol utilizationpathway components, one nucleic acid molecule (e.g., glycerol kinase)may be codon optimized, while the second nucleic acid molecule (e.g.,glycerol-3-phosphate dehydrogenase) is not codon optimized, or viceversa. Alternatively, all nucleic acids encoding multi-carbon substrate(e.g., glycerol) utilization pathway components may be codon optimized.

Transformation Methods

Any of the recombinant methanotrophic bacteria described herein may betransformed to comprise at least one exogenous nucleic acid to providethe host bacterium with a new or enhanced activity (e.g., enzymaticactivity) or may be genetically modified to remove or substantiallyreduce an endogenous gene function using a variety of methods known inthe art.

Transformation refers to the transfer of a nucleic acid (e.g., exogenousnucleic acid) into the genome of a host bacterium, resulting ingenetically stable inheritance. Host bacteria containing the transformednucleic acid molecules are referred to as “non-naturally occurring” or“recombinant” or “transformed” or “transgenic” bacteria.

Expression systems and expression vectors useful for the expression ofheterologous nucleic acids in methanotrophic bacteria are known.

Electroporation of C1 metabolizing bacteria has been previouslydescribed in Toyama et al., 1998, FEMS Microbiol. Lett. 166:1-7; Kim andWood, 1997, Appl. Microbiol. Biotechnol. 48:105-108; Yoshida et al.,2001, Biotechnol. Lett. 23:787-791, and US2008/0026005.

Bacterial conjugation, which refers to a particular type oftransformation involving direct contact of donor and recipient cells, ismore frequently used for the transfer of nucleic acids into C1metabolizing bacteria. Bacterial conjugation involves mixing “donor” and“recipient” cells together in close contact with each other. Conjugationoccurs by formation of cytoplasmic connections between donor andrecipient bacteria, with unidirectional transfer of newly synthesizeddonor nucleic acid molecules into the recipient cells. A recipient in aconjugation reaction is any cell that can accept nucleic acids throughhorizontal transfer from a donor bacterium. A donor in a conjugationreaction is a bacterium that contains a conjugative plasmid, conjugativetransposon, or mobilized plasmid. The physical transfer of the donorplasmid can occur through a self-transmissible plasmid or with theassistance of a “helper” plasmid. Conjugations involving C1 metabolizingbacteria have been previously described in Stolyar et al., 1995,Mikrobiologiya 64:686-691; Motoyama et al., 1994, Appl. Micro. Biotech.42:67-72; Lloyd et al., 1999, Archives of Microbiology 171:364-370; andOdom et al., PCT Publication WO 02/18617; Ali et al., 2006, Microbiol.152:2931-2942.

Expression of heterologous nucleic acids in C1 metabolizing bacteria isknown in the art (see, e.g., U.S. Pat. No. 6,818,424, US2003/0003528).Mu transposon based transformation of methylotrophic bacteria has beendescribed (Akhverdyan et al., 2011, Appl. Microbiol. Biotechnol.91:857-871). A mini-Tn7 transposon system for single and multicopyexpression of heterologous genes without insertional inactivation ofhost genes in Methylobacterium has been described (US2008/0026005).

Various methods for inactivating, knocking-out, or deleting endogenousgene function in C1 metabolizing bacteria may be used. Allelic exchangeusing suicide vectors to construct deletion/insertional mutants in slowgrowing C1 metabolizing bacteria have also been described in Toyama andLidstrom, 1998, Microbiol. 144:183-191; Stolyar et al., 1999, Microbiol.145:1235-1244; Ali et al., 2006, Microbiology 152:2931-2942; Van Dien etal., 2003, Microbiol. 149:601-609.

Suitable homologous or heterologous promoters for high expression ofexogenous nucleic acids may be utilized. For example, U.S. Pat. No.7,098,005 describes the use of promoters that are highly expressed inthe presence of methane or methanol for heterologous gene expression inC1 metabolizing bacteria. Additional promoters that may be used includedeoxy-xylulose phosphate synthase methanol dehydrogenase operon promoter(Springer et al., 1998, FEMS Microbiol. Lett. 160:119-124); the promoterfor PHA synthesis (Foellner et al. 1993, Appl. Microbiol. Biotechnol.40:284-291); or promoters identified from native plasmid inmethylotrophs (EP296484). Non-native promoters include the lac operonPlac promoter (Toyama et al., 1997, Microbiology 143:595-602) or ahybrid promoter such as Ptrc (Brosius et al., 1984, Gene 27:161-172). Incertain embodiments, promoters or codon optimization are used for highconstitutive expression of exogenous nucleic acids encoding glycerolutilization pathway enzymes in host methanotrophic bacteria. Regulatedexpression of an exogenous nucleic acid in the host methanotrophicbacterium may also be utilized. In particular, regulated expression ofexogenous nucleic acids encoding glycerol utilization enzymes may bedesirable to optimize growth rate of the non-naturally occurringmethanotrophic bacteria. It is possible that in the absence of glycerol(e.g., during growth on methane as sole carbon source), for the glycerolutilization pathway to run in reverse, resulting in secretion ofglycerol from the bacteria, thereby lowering growth rate. Controlledexpression of nucleic acids encoding glycerol utilization pathwayenzymes in response to the presence of glycerol may optimize bacterialgrowth in a variety of carbon source conditions. For example, aninducible/regulatable system of recombinant protein expression inmethylotrophic and methanotrophic bacteria as described inUS2010/0221813 may be used. Regulation of glycerol utilization genes inbacteria is well established (Schweizer and Po, 1996, J. Bacteriol.178:5215-5221; Abram et al., 2008, Appl. Environ. Microbiol. 74:594-604;Darbon et al., 2002, Mol. Microbiol. 43:1039-1052; Weissenborn et al.,1992, J. Biol. Chem. 267:6122-6131). Glycerol utilization regulatoryelements may also be introduced or inactivated in host methanotrophicbacteria for desired expression levels of exogenous nucleic acidmolecules encoding glycerol utilization pathway enzymes.

Methods of screening are disclosed in Brock, supra. Selection methodsfor identifying allelic exchange mutants are known in the art (see,e.g., U.S. Patent Publication No. 2006/0057726, Stolyar et al., 1999,Microbiol. 145:1235-1244; and Ali et al., 2006, Microbiology152:2931-2942.

Culture Methods

A variety of culture methodologies may be used for the recombinantmethanotrophic bacteria described herein. For example, methanotrophicbacteria may be grown by batch culture and continuous culturemethodologies. In certain embodiments, the cultures are grown in acontrolled culture unit, such as a fermentor, bioreactor, hollow fibermembrane bioreactor, or the like.

A classical batch culturing method is a closed system where thecomposition of the media is set at the beginning of the culture and notsubject to external alterations during the culture process. Thus, at thebeginning of the culturing process, the media is inoculated with thedesired methanotrophic bacteria and growth or metabolic activity ispermitted to occur without adding anything to the system. Typically,however, a “batch” culture is batch with respect to the addition ofcarbon source and attempts are often made at controlling factors such aspH and oxygen concentration. In batch systems, the metabolite andbiomass compositions of the system change constantly up to the time theculture is terminated. Within batch cultures, cells moderate through astatic lag phase to a high growth logarithmic phase and finally to astationary phase where growth rate is diminished or halted. Ifuntreated, cells in the stationary phase will eventually die. Cells inlogarithmic growth phase are often responsible for the bulk productionof end product or intermediate in some systems. Stationary orpost-exponential phase production can be obtained in other systems.

The Fed-Batch system is a variation on the standard batch system.Fed-Batch culture processes comprise a typical batch system with themodification that the substrate is added in increments as the cultureprogresses. Fed-Batch systems are useful when catabolite repression isapt to inhibit the metabolism of the cells and where it is desirable tohave limited amounts of substrate in the media. Measurement of theactual substrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measureable factors,such as pH, dissolved oxygen, and the partial pressure of waste gasessuch as CO₂. Batch and Fed-Batch culturing methods are common and knownin the art (see, e.g., Thomas D. Brock, Biotechnology: A Textbook ofIndustrial Microbiology, 2^(nd) Ed. (1989) Sinauer Associates, Inc.,Sunderland, Mass.; Deshpande, 1992, Appl. Biochem. Biotechnol. 36:227,herein incorporated by reference in its entirety).

Continuous cultures are “open” systems where a defined culture media isadded continuously to a bioreactor and an equal amount of conditionedmedia is removed simultaneously for processing. Continuous culturesgenerally maintain the cells at a constant high liquid phase densitywhere cells are primarily in logarithmic phase growth. Alternatively,continuous culture may be practiced with immobilized cells where carbonand nutrients are continuously added and valuable products, by-products,and waste products are continuously removed from the cell mass. Cellimmobilization may be performed using a wide range of solid supportscomposed of natural and/or synthetic materials.

Continuous or semi-continuous culture allows for the modulation of onefactor or any number of factors that affect cell growth or end productconcentration. For example, one method will maintain a limited nutrient,such as the carbon source or nitrogen level, at a fixed rate and allowall other parameters to modulate. In other systems, a number of factorsaffecting growth can be altered continuously while the cellconcentration, measured by media turbidity, is kept constant. Continuoussystems strive to maintain steady state growth conditions and thus thecell loss due to media being drawn off must be balanced against the cellgrowth rate in the culture. Methods of modulating nutrients and growthfactors for continuous culture processes, as well as techniques formaximizing the rate of product formation, are well known in the art, anda variety of methods are detailed by Brock, supra.

Culture media must contain suitable carbon substrates for themethanotrophic bacteria. A culture media may comprise a selectedmulti-carbon substrate (e.g., glycerol) as the sole carbon source forthe non-naturally occurring methanotrophic bacteria as described herein.Alternatively, a culture media may comprise two or more carbonsubstrates (mixed carbon substrates). Mixed carbon substrates maycomprise a mixture of a C1 substrate and a multi-carbon substrate. Forexample, the culture media may comprise a mixture of glycerol andmethane. Alternatively, mixed carbon substrates may comprise a mixtureof more than one multi-carbon substrate or more than one C1 substrate ora combination thereof. For cultures containing mixed carbon substrates,a selected multi-carbon substrate (e.g., glycerol) is used as a primarycarbon source by the recombinant methanotrophic bacteria as describedherein. A carbon source, whether a multi-carbon substrate alone or amixed composition, may be added to culture media initially, provided toculture media intermittently, or supplied continuously. Alternatively,recombinant methanotrophic bacteria may be initially grown in culturewith methane as a sole carbon source and then a multi-carbon substrateadded at a later time point to make a mixed carbon source or methane maybe substituted by a multi-carbon substrate.

Glycerol compositions added to the culture may be purified glycerol orcrude glycerol. Purified glycerol, the refined form used inpharmaceutical, food, and cosmetic industries, is at least 90%, 95%,96%, 97%, 98%, 99%, or 99.5% pure. Crude glycerol is a by-product ofbiodiesel production that contains approximately 50-60% of glycerol,12-16% of alkalies, especially in the form of alkali soaps andhydroxides, 15-18% of methyl esters, 8-12% of methanol, 2-3% water, andfurther components. Crude glycerol also contains a variety of elements,such as calcium, magnesium, phosphorus, or sulfur, originating from theprimary oil. Larger quantities of sodium or potassium are also present,derived from the catalyst. Purified or crude glycerol may be addeddirectly to the culture. Alternatively, impurities may be removed fromcrude glycerol by conventional separation techniques prior to additionto culture in order to increase the concentration of glycerol insolution to at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

Methods for Growing Recombinant Methanotrophic Bacteria

In certain embodiments, methods for growing recombinant methanotrophicbacteria are provided, comprising: culturing any of the embodiments ofrecombinant methanotrophic bacteria disclosed herein in the presence ofa multi-carbon substrate, wherein the multi-carbon substrate is used asa primary carbon source by the recombinant obligate methanotrophicbacteria or wherein the multi-carbon substrate is used as a sole carbonsource by the recombinant facultative methanotrophic bacteria. Amulti-carbon substrate may include glucose, acetate, lactate, arabinose,citrate, succinate, and glycerol. In certain embodiments, a selectedmulti-carbon substrate is used as a sole carbon source. In certainembodiments, the multi-carbon substrate is used as a sole carbon sourceby the recombinant obligate methanotrophic bacteria.

In certain embodiments wherein the multi-carbon substrate is glucose,the recombinant methanotrophic bacteria include at least one exogenousnucleic acid encoding a glucose transporter.

In certain embodiments wherein the multi-carbon substrate is acetate,the recombinant methanotrophic bacteria include at least one exogenousnucleic acid encoding an acetate transporter. In further embodiments,the recombinant methanotrophic bacterium is further modified tooverexpress acetyl-CoA synthase.

In certain embodiments wherein the multi-carbon substrate is lactate,the recombinant methanotrophic bacteria include an exogenous nucleicacid encoding a lactate transporter and an exogenous nucleic acidencoding a lactate dehydrogenase.

In certain embodiments wherein the multi-carbon substrate is arabinose,the recombinant methanotrophic bacteria include an exogenous nucleicacid encoding an L-arabinose isomerase (e.g., AraA), an exogenousnucleic acid encoding an L-ribulose kinase (e.g., AraB), an exogenousnucleic acid encoding an L-ribulose-5-phosphate epimerase (e.g., AraD).An arabinose transporter includes AraE, AraFGH, and AraP.

In certain embodiments wherein the multi-carbon substrate is citrate,the recombinant methanotrophic bacteria include an exogenous nucleicacid encoding a citrate transporter.

In certain embodiments wherein the multi-carbon substrate is succinate,the recombinant methanotrophic bacteria include an exogenous nucleicacid encoding a succinate transporter.

In certain embodiments wherein the multi-carbon substrate is glycerol,the recombinant methanotrophic bacteria include at least two exogenousnucleic acids encoding glycerol utilization pathway components. The atleast two exogenous nucleic acids encoding glycerol utilization pathwaycomponents may comprise glycerol kinase and glycerol-3-phosphatedehydrogenase. In a specific embodiment, glycerol kinase is GlpK andglycerol-3-phosphate dehydrogenase is GlpD. In another specificembodiment, glycerol kinase comprises an amino acid sequence of SEQ IDNO:22 and glycerol-3-phosphate dehydrogenase comprises an amino acidsequence of SEQ ID NO:43.

In certain embodiments, the recombinant methanotrophic bacteria includesthree exogenous nucleic acids encoding glycerol utilization pathwaycomponents. The three exogenous nucleic acids encoding glycerolutilization pathway components may comprise glycerol uptake facilitator,glycerol kinase, and glycerol-3-phosphate dehydrogenase. In a specificembodiment, glycerol uptake facilitator is GlpF, glycerol kinase isGlpK, and glycerol-3-phosphate dehydrogenase is GlpD. In anotherspecific embodiment, glycerol uptake facilitator comprises an amino acidsequence of SEQ ID NO:1, glycerol kinase comprises an amino acidsequence of SEQ ID NO:22, and glycerol-3-phosphate dehydrogenasecomprises an amino acid sequence of SEQ ID NO:43.

Obligate methanotrophic bacteria for use according to any of theembodied methods disclosed herein include Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, orMethanomonas. Exemplary obligate methanotrophic bacteria include:Methylococcus capsulatus Bath strain, Methylomonas 16a (ATCC PTA 2402),Methylosinus trichosporium OB3b (NRRL B-11,196), Methylosinus sporium(NRRL B-11,197), Methylocystis parvus (NRRL B-11,198), Methylomonasmethanica (NRRL B-11,199), Methylomonas albus (NRRL B-11,200),Methylobacter capsulatus (NRRL B-11,201), Methylomonas sp AJ-3670 (FERMP-2400), Methylacidiphilum infernorum( ), and Methylomicrobiumalcaliphilum 20Z. Exemplary facultative methanotrophic bacteria for useaccording to any of the embodied methods disclosed herein includeMethylocella silvestris, Methylocella palustris, Methylocella tundra,Methylocystis daltona, Methylocystis bryophila, Methylobacteriumorganophilum (ATCC 27,886), and Methylocapsa aurea.

In certain embodiments, one or more exogenous nucleic acids encoding amulti-carbon substrate utilization pathway component is codon optimizedfor high expression in methanotrophic bacteria.

Measuring Utilization of Multi-Carbon Substrates

Utilization of a selected multi-carbon substrate as a sole carbon sourcemay be measured by determining growth rate, biomass yield, or increasein cell density using standard methods known in the art during cultureof recombinant bacteria with the selected multi-carbon substrate as thesole carbon source.

Measuring Glycerol Utilization

Glycerol utilization by recombinant methanotrophic bacteria describedherein may be determined by various methods known in the art. Forexample, enzyme activity assays of glycerol utilization pathway enzymes,e.g., glycerol kinase and glycerol-3-phosphate dehydrogenase, have beenpreviously described (Rawls et al., 2011, J. Bacteriol. 193:4469-4476;Rittman et al., 2008, Applied Environmental Microbiol. 74:6216-6222;Darbon et al., 1999, Microbiol. 145:3205-3212; Charrier et al., 1997, J.Biol. Chem. 272:14166-14174).

Dihydroxyacetone phosphate (DHAP) levels may also be assayed via acoupled enzyme system: reduction with NADH-consumingglycerol-3-phosphate dehydrogenase enables determination of DHAP bymeasuring NADH concentration with UV spectroscopy (WO2007/003574).

The growth rate or biomass yield of recombinant methanotrophic usingglycerol as a carbon source may also be measured as an indicator ofglycerol utilization (Rittman et al., 2008, Applied EnvironmentalMicrobiol. 74:6216-6222; Muraka et al., 2008, Applied EnvironmentalMicrobiol. 74:1124-1135).

Glycerol depletion from the culture medium may also be measured overtime. At various time points, aliquots of culture broth containingglycerol and non-naturally occurring methanotrophic bacteria describedherein may be withdrawn, centrifuged, filtered, and analyzed byhigh-performance liquid chromatography to determine consumption ofglycerol during bacterial growth in the medium (Rawls et al., 2011, J.Bacteriol. 193:4469-4476; Gonzalez et al., 2008, Metabolic Engineering10:234-245; Rittman et al., 2008, Applied Environmental Microbiol.74:6216-6222). Alternatively, radio-labeled glycerol substrate may beadded to the culture media, and after different time points,radioactivity remaining in the culture or incorporated into bacteria maybe measured (Sher et al., 2004, FEMS Microbiol. Lett. 232:211-215;Muraka et al. 2008, Applied Environmental Microbiol. 74:1124-1135).Glycerol concentration in bacterial cell culture supernatant may also bemeasured at various time points using a free glycerol assay kit (see,e.g., Product #FG0100 from Sigma, St. Louis, Mo. or Product #ab65337from Abcam, Cambridge Mass.).

Measuring Glucose Utilization

Glucose consumption may be measured by sampling culture medium over timefor glucose or its corresponding fermentation by-products using HPLC(see, e.g., Garcia Sanchez et al., 2010, Biotechnology for Biofuels3:13). Coupled enzyme assays, such as the one described in U.S. Pat. No.4,490,465 or Amplex® Red Glucose/Glucose Oxidase Assay Kit (Invitrogen,Catalog #A22189), may be used to determine the amount of glucose insolution.

Measuring Acetate Utilization

Acetate agar may be used to test recombinant methanotrophic bacteria'sability to utilize acetate. The medium contains sodium acetate as thesole carbon source and inorganic ammonium salts as the sole nitrogensource. Bacterial growth is indicative of acetate utilization. When thebacteria metabolize acetate, the ammonium salts are broken down toammonia, increasing alkalinity. The resulting increase in pH turns thebromothymol blue indicator in the medium from green to blue (see, e.g.,Weyant et al. 1995, Identification of Unusual Pathogenic Gram-NegativeAerobic and Facultatively Anaerobic Bacteria, 2^(nd) ed., pp. 6-7,Williams & Wilkins, Baltimore, Md.).

Acetate utilization may also be measuring using a method as described inDedysh et al. (2005, J. Bacteriol. 187:4665-4670). Briefly, recombinantmethanotrophic bacteria are grown in basal salts medium DNMS (dilutenitrate mineral salts, pH 5.8) with sodium acetate at 0.04% wt/vol.Cultures are grown to an OD600 of >0.1 from an inoculation of <0.001 at25° C. on a rotary shaker at 120 rpm. Samples are taken daily fordetermination of acetate concentrations, direct microscopic cell counts,OD600, and DNA extraction and quantitative real-time PCR todetermination acetate utilization and bacterial cell growth. Acetate ismeasured on a Sykam high-performance liquid chromatography system with arefraction index detector. Measurement of growth yield and carbonconversion efficiency on acetate substrate has also been described inU.S. Patent Publication 2012/0034594. Assay kits for detecting acetateare commercially available (e.g., Catalog #K-ACETRM from Megazyme,Wicklow, Ireland)

Measuring Lactate Utilization

Lactate uptake assays may be used to measure lactate utilization.Briefly, radiolabelled lactate is added to cell culture and afterdifferent time intervals, samples are taken, filtered, washed, andcounted on a scintillator (see, e.g., Nunez et al., 2001, Microbiol.147:1069-1077; Exley et al., 2007, Infect. Immun. 75:1318-1324).

Lactate utilization may also be measuring by testing for conversion oflactate to pyruvate by lactate dehydrogenase and performing kineticanalysis (see, e.g., Garvie, 1980, Microbiol. Rev. 44:106-139; Brown andWhiteley, 2009, PLoS One 4:e7864; Futai, 1973, Biochemistry12:2468-2474; Futai and Kimura, 1977, J. Biol. Chem. 252:5820-5827; Kohnand Kaback, 1973, J. Biol. Chem. 248:7012-7017; Molinari and Lara, 1960,Biochem. J. 75:57-65; Kline and Mahler, 1965, Ann. N.Y. Acad. Sci.119:905-919). Coupled enzyme assays, such as the one described in U.S.Pat. No. 4,490,465 may be used to determine the amount of lactate insolution.

Measuring Arabinose Utilization

Arabinose utilization may be tested using an arabinose uptake assay(see, e.g. Subtil and Boles, 2011, Biotechnology for Biofuels, 4:38;U.S. Patent Publication 2012/0129241; Poysti et al., 2001, Microbiol.153:727-736). Briefly, recombinant methanotrophic bacteria are incubatedwith radiolabeled arabinose, and after different time intervals, cellsare collected. The suspension is immediately filtered, washed, andradioactivity of the filtrate is measured in a scintillation counter.

Alternatively, arabinose utilization may be measured by determiningL-arabinose isomerase (e.g., AraA), L-ribulose kinase (e.g., AraB), orL-ribulose 5-phosphate epimerase (e.g., AraD) enzyme activities (see,e.g., Sedlak and Ho, 2001, Enzyme and Microbial Technol. 28:16-24;Shamanna and Sanderson, 1979, J. Bacteriol. 139:64-70; Lee et al., 1968,J. Biol. Chem. 243:4700-4705).

Measuring Citrate Utilization

Citrate utilization as a sole carbon source may be detecting using amedium containing sodium citrate, a pH indicator (e.g., bromothymolblue), and inorganic ammonium salts as a sole nitrogen source. Duringits metabolism, citrate is converted to oxaloacetate and acetate.Production of NaHCO₃ and NH₃ from the use of sodium citrate and ammoniumsalts results in alkaline conditions, which is detected by a change ofthe medium's color from green to blue.

Measuring Succinate Utilization

Succinate utilization may be measuring by a succinate uptake by cellsduring culture using ¹⁴-labeled succinate (see, e.g., Weiss, 1970 J.Bacteriol. 101:133-137; Gutowski and Rosenberg, 1975, Biochem. J.152:647-654; or Glenn et al., 1980, Microbiol. 119:267-271).

EXAMPLES Example 1 Recombinant Methylosinus trichosporium Engineered toGrow on Glycerol

Preparation of NMS Media.

MgSO₄•7H₂O 1.0 g CaCl₂•6H₂O 0.20 g Chelated Iron Solution (see below)2.0 ml KNO₃ 1.0 g Trace Element Solution (see below) 0.5 ml KH₂PO₄ 0.272g Na2HPO₄•12H₂O 0.717 g Purified Agar (e.g., Oxoid L28) 12.5 g Distilleddeionized water 1.0 L

Adjust pH to 6.8. Autoclave at 121° C. for 15 minutes.

Chelated Iron Solution:

Ferric (III) ammonium citrate* 0.1 g EDTA, sodium salt 0.2 g HCl(concentrated) 0.3 ml Distilled deionized water 100.0 ml *0.5 g ofFerric (III) chloride may be substituted.

Use 2.0 ml of this chelated iron solution per liter of final medium.

Trace Element Solution:

EDTA 500.0 mg FeSO₄•7H₂O 200.0 mg ZnSO₄•7H₂O 10.0 mg MnCl₂•4H₂O 3.0 mgH₃BO₃ 30.0 mg CoCl₂•6H₂O 20.0 mg CaCl₂•2H₂O 1.0 mg NiCl₂•6H₂O 2.0 mgNa₂MoO₄•2H₂O 3.0 mg Distilled water 1.0 L Autoclave at 121° C. for 15minutes.

Growth and Conjugations. The procedure for conjugating plasmids from E.coli into methanotrophs was based on the method developed by Martin, H.& Murrell, J. C. (1995). Methane monooxygenase mutants of Methylosinustrichosporium constructed by marker exchange mutagenesis. FEMSMicrobiol. Lett. 127:243-248.

Briefly, a mobilizing plasmid to be conjugated was first transformedinto E. coli S17-1 using standard electroporation methods.Transformation was confirmed by selection of kanamycin-resistantcolonies on LB-agar containing 20 ug/mL kanamycin. Transformed colonieswere inoculated into LB media containing 20 ug/mL kanamycin and shakenovernight at 37° C. A 10 mL aliquot of the overnight culture was thencollected on a sterile 47 mm nitrocellulose filter (0.2 mm pore size).The E. coli donor strain was washed on the filter with 50 mL sterile NMSmedia to remove residual media and antibiotic.

In parallel, a sample of the M. trichosporium OB3b recipient strain wasinoculated into 100 mL serum bottles containing 20-50 mL NMS media. Theheadspace of the bottles was then flushed with a 1:1 mixture of oxygenand methane, and the bottles were sealed with butyl rubber septa andcrimped. The bottles were shaken continuously in a 30° C. incubatoruntil reaching an OD600 of approximately 0.3. The cells were thencollected on the same filter as the E. coli donor strain. The filter wasagain washed with 50 mL of sterile NMS media. The filter was placed(cells up) on an NMS agar plate containing 0.02% (w/v) proteose peptoneand incubated for 24 h at 30° C. in the presence of methane and oxygen.After 24 h, cells were resuspended in 10 mL sterile (NMS) medium beforebeing concentrated by centrifugation. The pellet was resuspended in 1 mLsterile NMS media. Aliquots (100 μl) were spread onto NMS agar platescontaining 10 ug/mL kanamycin.

The plates were incubated in sealed chambers containing a 1:1 mixture ofmethane and oxygen maintained at 30° C. The gas mixture was replenishedevery 2 days until colonies formed, typically after 7-14 days. Colonieswere streaked onto NMS plates containing kanamycin to confirm kanamycinresistance as well as to further isolate transformed methanotroph cellsfrom residual E. coli donor cells.

Introduction of Glycerol Utilization Pathway. Nucleic acids encodingGlpK, GlpD, and GlpF from E. coli were codon optimized for expression inMethylosinus trichosporium. The codon optimized nucleic acids encodingGlpK, GlpD, and GlpF are synthesized as an operon (SEQ ID NO:95; seeTable 14 for components of operon) under control of an mdh promoter withappropriate intergenic regions (CAPITALIZED sequence) incorporatingribosome binding sequences.

TABLE 14Glycerol Utilization Pathway Operon Codon Optimized for Methylosinustrichosporium SEQ ID NO: # Gene Name Nucleotide Sequence SEQ ID MDHTTTGCCTCGATCGGCGGTCCTTGTGACAGGGAG NO: 89 promoterATATTCCCGACGGATCCGGGGCATTCGAGCGGA ACCGCCCGCCGTGGGAGTTTTTCCAGCGAGCATTCGAGAGTTTTTCAAGGCGGCTTCGAGGGGTTA TTCCGTAACGCCGCCGACATGATCTGTCCCAGAATCTCCGCCGCTGTTCGTAGAGCGCCGATGCAG GGTCGGCATCAATCATTCTTGGAGGAGACAC SEQ IDGlpK atgaccgagaagaagtatatcgtcgcgctggaccagggcaccacctcgtcgc NO: 90gcgcggtcgtcatggaccatgacgcgaacatcatctcggtctcgcagcgcgagttcgagcagatctatccgaagccgggctgggtcgagcatgacccgatggagatctgggcgacccagtcgtcgaccctggtcgaggtcctggcgaaggcggacatctcgtcggaccagatcgcggcgatcggcatcaccaaccagcgcgagaccaccatcgtctgggagaaggagaccggcaagccgatctataacgcgatcgtctggcagtgccgccgcaccgcggagatctgcgagcatctgaagcgcgacggcctggaggactatatccgctcgaacaccggcctggtcatcgacccgtatttctcgggcaccaaggtcaagtggatcctggaccatgtcgagggctcgcgcgagcgcgcgcgccgcggcgagctgctgttcggcaccgtcgacacctggctgatctggaagatgacccagggccgcgtccatgtcaccgactataccaacgcgtcgcgcaccatgctgttcaacatccataccctggactgggacgacaagatgctggaggtcctggacatcccgcgcgagatgctgccggaggtccgccgctcgtcggaggtctatggccagaccaacatcggcggcaagggcggcacccgcatcccgatctcgggcatcgcgggcgaccagcaggcggcgctgttcggccagctgtgcgtcaaggagggcatggcgaagaacacctatggcaccggctgcttcatgctgatgaacaccggcgagaaggcggtcaagtcggagaacggcctgctgaccaccatcgcgtgcggcccgaccggcgaggtcaactatgcgctggagggcgcggtcttcatggcgggcgcgtcgatccagtggctgcgcgacgagatgaagctgatcaacgacgcgtatgactcggagtatttcgcgaccaaggtccagaacaccaacggcgtctatgtcgtcccggcgttcaccggcctgggcgcgccgtattgggacccgtatgcgcgcggcgcgatcttcggcctgacccgcggcgtcaacgcgaaccatatcatccgcgcgaccctggagtcgatcgcgtatcagacccgcgacgtcctggaggcgatgcaggcggactcgggcatccgcctgcatgcgctgcgcgtcgacggcggcgcggtcgcgaacaacttcctgatgcagttccagtcggacatcctgggcacccgcgtcgagcgcccggaggtccgcgaggtcaccgcgctgggcgcggcgtatctggcgggcctggcggtcggcttctggcagaacctggacgagctgcaggagaaggcggtcatcgagcgcgagttccgcccgggcatcgagaccaccgagcgcaactatcgctatgcgggctggaagaaggcggtcaagcgcgcgatggcgtgggag gagcatgactga SEQ IDIntergenic TCATTCTTGGAGGAGACAC NO: 91 region SEQ ID GlpDatggagaccaaggacctgatcgtcatcggcggcggcatcaacggcgcgggc NO: 92atcgcggcggacgcggcgggccgcggcctgtcggtcctgatgctggaggcgcaggacctggcgtgcgcgacctcgtcggcgtcgtcgaagctgatccatggcggcctgcgctttctggagcattatgagttccgcctggtctcggaggcgctggcggagcgcgaggtcctgctgaagatggcgccgcatatcgcgttcccgatgcgcttccgcctgccgcatcgcccgcatctgcgcccggcgtggatgatccgcatcggcctgttcatgtatgaccatctgggcaagcgcacctcgctgccgggctcgaccggcctgcgcttcggcgcgaactcggtcctgaagccggagatcaagcgcggcttcgagtattcggactgctgggtcgacgacgcgcgcctggtcctggcgaacgcgcagatggtcgtccgcaagggcggcgaggtcctgacccgcacccgcgcgacctcggcgcgccgcgagaacggcctgtggatcgtcgaggcggaggacatcgacaccggcaagaagtattcgtggcaggcgcgcggcctggtcaacgcgaccggcccgtgggtcaagcagttcttcgacgacggcatgcatctgccgtcgccgtatggcatccgcctgatcaagggctcgcatatcgtcgtcccgcgcgtccatacccagaagcaggcgtatatcctgcagaacgaggacaagcgcatcgtcttcgtcatcccgtggatggacgagttctcgatcatcggcaccaccgacgtcgagtataagggcgacccgaaggcggtcaagatcgaggagtcggagatcaactatctgctgaacgtctataacacccatttcaagaagcagctgtcgcgcgacgacatcgtctggacctattcgggcgtccgcccgctgtgcgacgacgagtcggactcgccgcaggcgatcacccgcgactataccctggacatccatgacgagaacggcaaggcgccgctgctgtcggtcttcggcggcaagctgaccacctatcgcaagctggcggagcatgcgctggagaagctgaccccgtattatcagggcatcggcccggcgtggaccaaggagtcggtcctgccgggcggcgcgatcgagggcgaccgcgacgactatgcggcgcgcctgcgccgccgctatccgttcctgaccgagtcgctggcgcgccattatgcgcgcacctatggctcgaactcggagctgctgctgggcaacgcgggcaccgtctcggacctgggcgaggacttcggccatgagttctatgaggcggagctgaagtatctggtcgaccatgagtgggtccgccgcgcggacgacgcgctgtggcgccgcaccaagcagggcatgtggctgaacgcggaccagcagtcgcgcgtctcgcagtggctggtcgagtatacccagcagcgcctgtcgctg gcgtcgtga SEQ IDIntergenic TCATTCTTGGAGGAGACAC NO: 93 region SEQ ID GlpFatgtcgcagacctcgaccctgaagggccagtgcatcgcggagttcctgggca NO: 94ccggcctgctgatcttcttcggcgtcggctgcgtcgcggcgctgaaggtcgcgggcgcgtcgttcggccagtgggagatctcggtcatctggggcctgggcgtcgcgatggcgatctatctgaccgcgggcgtctcgggcgcgcatctgaacccggcggtcaccatcgcgctgtggctgttcgcgtgcttcgacaagcgcaaggtcatcccgttcatcgtctcgcaggtcgcgggcgcgttctgcgcggcggcgctggtctatggcctgtattataacctgttcttcgacttcgagcagacccatcatatcgtccgcggctcggtcgagtcggtcgacctggcgggcaccttctcgacctatccgaacccgcatatcaacttcgtccaggcgttcgcggtcgagatggtcatcaccgcgatcctgatgggcctgatcctggcgctgaccgacgacggcaacggcgtcccgcgcggcccgctggcgccgctgctgatcggcctgctgatcgcggtcatcggcgcgtcgatgggcccgctgaccggcttcgcgatgaacccggcgcgcgacttcggcccgaaggtcttcgcgtggctggcgggctggggcaacgtcgcgttcaccggcggccgcgacatcccgtatttcctggtcccgctgttcggcccgatcgtcggcgcgatcgtcggcgcgttcgcgtatcgcaagctgatcggccgccatctgccgtgcgacatctgcgtcgtcgaggagaaggagaccaccaccccgtcggagcagaaggc gtcgctgtga SEQ IDGlycerol tttgcctcgatcggcggtccttgtgacagggagatattcccgacggatccggg NO: 95Utilization gcattcgagcggaaccgcccgccgtgggagtttttccagcgagcattcgaga Pathwaygtttttcaaggcggcttcgaggggttattccgtaacgccgccgacatgatctgtc Operonccagaatctccgccgctgttcgtagagcgccgatgcagggtcggcatcaatcattcttggaggagacacatgaccgagaagaagtatatcgtcgcgctggaccagggcaccacctcgtcgcgcgcggtcgtcatggaccatgacgcgaacatcatctcggtctcgcagcgcgagttcgagcagatctatccgaagccgggctgggtcgagcatgacccgatggagatctgggcgacccagtcgtcgaccctggtcgaggtcctggcgaaggcggacatctcgtcggaccagatcgcggcgatcggcatcaccaaccagcgcgagaccaccatcgtctgggagaaggagaccggcaagccgatctataacgcgatcgtctggcagtgccgccgcaccgcggagatctgcgagcatctgaagcgcgacggcctggaggactatatccgctcgaacaccggcctggtcatcgacccgtatttctcgggcaccaaggtcaagtggatcctggaccatgtcgagggctcgcgcgagcgcgcgcgccgcggcgagctgctgttcggcaccgtcgacacctggctgatctggaagatgacccagggccgcgtccatgtcaccgactataccaacgcgtcgcgcaccatgctgttcaacatccataccctggactgggacgacaagatgctggaggtcctggacatcccgcgcgagatgctgccggaggtccgccgctcgtcggaggtctatggccagaccaacatcggcggcaagggcggcacccgcatcccgatctcgggcatcgcgggcgaccagcaggcggcgctgttcggccagctgtgcgtcaaggagggcatggcgaagaacacctatggcaccggctgcttcatgctgatgaacaccggcgagaaggcggtcaagtcggagaacggcctgctgaccaccatcgcgtgcggcccgaccggcgaggtcaactatgcgctggagggcgcggtcttcatggcgggcgcgtcgatccagtggctgcgcgacgagatgaagctgatcaacgacgcgtatgactcggagtatttcgcgaccaaggtccagaacaccaacggcgtctatgtcgtcccggcgttcaccggcctgggcgcgccgtattgggacccgtatgcgcgcggcgcgatcttcggcctgacccgcggcgtcaacgcgaaccatatcatccgcgcgaccctggagtcgatcgcgtatcagacccgcgacgtcctggaggcgatgcaggcggactcgggcatccgcctgcatgcgctgcgcgtcgacggcggcgcggtcgcgaacaacttcctgatgcagttccagtcggacatcctgggcacccgcgtcgagcgcccggaggtccgcgaggtcaccgcgctgggcgcggcgtatctggcgggcctggcggtcggcttctggcagaacctggacgagctgcaggagaaggcggtcatcgagcgcgagttccgcccgggcatcgagaccaccgagcgcaactatcgctatgcgggctggaagaaggcggtcaagcgcgcgatggcgtgggaggagcatgactgatcattcttggaggagacacatggagaccaaggacctgatcgtcatcggcggcggcatcaacggcgcgggcatcgcggcggacgcggcgggccgcggcctgtcggtcctgatgctggaggcgcaggacctggcgtgcgcgacctcgtcggcgtcgtcgaagctgatccatggcggcctgcgctatctggagcattatgagttccgcctggtctcggaggcgctggcggagcgcgaggtcctgctgaagatggcgccgcatatcgcgttcccgatgcgcttccgcctgccgcatcgcccgcatctgcgcccggcgtggatgatccgcatcggcctgttcatgtatgaccatctgggcaagcgcacctcgctgccgggctcgaccggcctgcgcttcggcgcgaactcggtcctgaagccggagatcaagcgcggcttcgagtattcggactgctgggtcgacgacgcgcgcctggtcctggcgaacgcgcagatggtcgtccgcaagggcggcgaggtcctgacccgcacccgcgcgacctcggcgcgccgcgagaacggcctgtggatcgtcgaggcggaggacatcgacaccggcaagaagtattcgtggcaggcgcgcggcctggtcaacgcgaccggcccgtgggtcaagcagttcttcgacgacggcatgcatctgccgtcgccgtatggcatccgcctgatcaagggctcgcatatcgtcgtcccgcgcgtccatacccagaagcaggcgtatatcctgcagaacgaggacaagcgcatcgtcttcgtcatcccgtggatggacgagttctcgatcatcggcaccaccgacgtcgagtataagggcgacccgaaggcggtcaagatcgaggagtcggagatcaactatctgctgaacgtctataacacccattcaagaagcagctgtcgcgcgacgacatcgtctggacctattcgggcgtccgcccgctgtgcgacgacgagtcggactcgccgcaggcgatcacccgcgactataccctggacatccatgacgagaacggcaaggcgccgctgctgtcggtcttcggcggcaagctgaccacctatcgcaagctggcggagcatgcgctggagaagctgaccccgtattatcagggcatcggcccggcgtggaccaaggagtcggtcctgccgggcggcgcgatcgagggcgaccgcgacgactatgcggcgcgcctgcgccgccgctatccgttcctgaccgagtcgctggcgcgccattatgcgcgcacctatggctcgaactcggagctgctgctgggcaacgcgggcaccgtctcggacctgggcgaggacttcggccatgagttctatgaggcggagctgaagtatctggtcgaccatgagtgggtccgccgcgcggacgacgcgctgtggcgccgcaccaagcagggcatgtggctgaacgcggaccagcagtcgcgcgtctcgcagtggctggtcgagtatacccagcagcgcctgtcgctggcgtcgtgatcattcttggaggagacacatgtcgcagacctcgaccctgaagggccagtgcatcgcggagttcctgggcaccggcctgctgatcttcttcggcgtcggctgcgtcgcggcgctgaaggtcgcgggcgcgtcgttcggccagtgggagatctcggtcatctggggcctgggcgtcgcgatggcgatctatctgaccgcgggcgtctcgggcgcgcatctgaacccggcggtcaccatcgcgctgtggctgttcgcgtgcttcgacaagcgcaaggtcatcccgttcatcgtctcgcaggtcgcgggcgcgttctgcgcggcggcgctggtctatggcctgtattataacctgttcttcgacttcgagcagacccatcatatcgtccgcggctcggtcgagtcggtcgacctggcgggcaccttctcgacctatccgaacccgcatatcaacttcgtccaggcgttcgcggtcgagatggtcatcaccgcgatcctgatgggcctgatcctggcgctgaccgacgacggcaacggcgtcccgcgcggcccgctggcgccgctgctgatcggcctgctgatcgcggtcatcggcgcgtcgatgggcccgctgaccggcttcgcgatgaacccggcgcgcgacttcggcccgaaggtcttcgcgtggctggcgggctggggcaacgtcgcgttcaccggcggccgcgacatcccgtatttcctggtcccgctgttcggcccgatcgtcggcgcgatcgtcggcgcgttcgcgtatcgcaagctgatcggccgccatctgccgtgcgacatctgcgtcgtcgaggagaaggagaccaccaccccgtcggagcagaaggcgtcgctgtga

The synthetic operon is then cloned and transformed as described above.Transformation is confirmed by resistance of the cells to antibioticselection, and gene expression is confirmed by northern blot (to confirmRNA transcription), western blot, or ELISA methods (to confirm proteinexpression).

Growth on Glycerol as a Sole Carbon Source. Recombinant M. trichosporiumtransformed with a vector containing the synthetic operon encoding genesfor glycerol utilization are inoculated into 100 mL shake flaskscontaining 20-50 mL NMS media, 1% glycerol and 10 ug/mL kanamycin. Theflasks are then shaken continuously while being incubated at 30° C.Growth is confirmed by monitoring optical density of the culture overtime. Note that because glycerol is the only carbon source provided tothe cells, all cell mass produced must have been derived from glycerol.

Example 2 Recombinant Methylococcus capsulatus Bath Strain Engineered toGrow on Glycerol

Growth and Conjugations. The procedure for conjugating plasmids from E.coli into M. capsulatus was based on the method reported in Ali, H. &Murrell, J. C. (2009). Development and validation of promoter-probevectors for the study of methane monooxygenase gene expression inMethylococcus capsulatus Bath. Microbiology (2009), 155:761-771.

Briefly, a mobilizing plasmid to be conjugated was first transformedinto E. coli S17-1 using standard electroporation methods.Transformation was confirmed by selection of kanamycin-resistantcolonies on LB-agar containing 20 ug/mL kanamycin. Transformed colonieswere inoculated into LB media containing 20 ug/mL kanamycin and shakenovernight at 37° C. A 10 mL aliquot of the overnight culture was thencollected on a sterile 47 mm nitrocellulose filter (0.2 mm pore size).The E. coli donor strain was washed on the filter with 50 mL sterile NMSto remove residual media and antibiotic.

In parallel, a sample of the M. capsulatus recipient strain wasinoculated into 100 mL serum bottles containing 20-50 mL NMS media. Theheadspace of the bottles was then flushed with a 1:1 mixture of oxygenand methane, and the bottles were sealed with butyl rubber septa andcrimped. The bottles were shaken continuously in a 45° C. incubatoruntil reaching an OD₆₀₀ of approximately 0.3. The cells were thencollected on the same filter as the E. coli donor strain. The filter wasagain washed with 50 mL of sterile NMS media. The filter was placed(cells up) on an NMS agar plate containing 0.02% (w/v) proteose peptoneand incubated for 24 h at 37° C. in the presence of methane and oxygen.After 24 h, cells were resuspended in 10 mL sterile (NMS) medium beforebeing concentrated by centrifugation. The pellet was resuspended in 1 mLsterile NMS media. Aliquots (100 ul) were spread onto NMS agar platescontaining 10 ug/mL kanamycin.

The plates were incubated in sealed chambers containing a 1:1 mixture ofmethane and oxygen maintained at 45° C. The gas mixture was replenishedevery 2 days until colonies formed, typically after 7-14 days. Colonieswere streaked onto NMS plates containing kanamycin to confirm kanamycinresistance as well as to further isolate transformed methanotroph cellsfrom residual E. coli donor cells.

Introduction of Glycerol Utilization Pathway. Nucleic acid sequencesencoding GlpK, GlpD, and GlpF from E. coli were codon optimized forexpression in M. capsulatus. The codon optimized nucleic acids encodingGlpK, GlpD, and GlpF are synthesized as an operon (SEQ ID NO:102; seeTable 15 for components of operon) under control of an mdh promoter withappropriate intergenic regions (CAPITALIZED sequence) incorporatingribosome binding sequences.

TABLE 15Glycerol Utilization Pathway Operon Codon Optimized for M. capsulatusSEQ ID Gene NO: # Name Nucleotide Sequence SEQ ID MDHTTTGCCTCGATCGGCGGTCCTTGTGACAGGGAG NO: 96 promoterATATTCCCGACGGATCCGGGGCATTCGAGCGG AACCGCCCGCCGTGGGAGTTTTTCCAGCGAGCATTCGAGAGTTTTTCAAGGCGGCTTCGAGGGGTT ATTCCGTAACGCCGCCGACATGATCTGTCCCAGAATCTCCGCCGCTGTTCGTAGAGCGCCGATGCA GGGTCGGCATCAATCATTCTTGGAGGAGACACSEQ ID GlpK atgaccgagaagaagtacatcgtcgccctggaccagggcaccaccagcagc NO: 97cgcgccgtcgtcatggaccacgacgccaacatcatcagcgtcagccagcgcgagttcgagcagatctacccgaagccgggctgggtcgagcacgacccgatggagatctgggccacccagagcagcaccctggtcgaggtcctggccaaggccgacatcagcagcgaccagatcgccgccatcggcatcaccaaccagcgcgagaccaccatcgtctgggagaaggagaccggcaagccgatctacaacgccatcgtctggcagtgccgccgcaccgccgagatctgcgagcacctgaagcgcgacggcctggaggactacatccgcagcaacaccggcctggtcatcgacccgtacttcagcggcaccaaggtcaagtggatcctggaccacgtcgagggcagccgcgagcgcgcccgccgcggcgagctgctgttcggcaccgtcgacacctggctgatctggaagatgacccagggccgcgtccacgtcaccgactacaccaacgccagccgcaccatgctgttcaacatccacaccctggactgggacgacaagatgctggaggtcctggacatcccgcgcgagatgctgccggaggtccgccgcagcagcgaggtctacggccagaccaacatcggcggcaagggcggcacccgcatcccgatcagcggcatcgccggcgaccagcaggccgccctgttcggccagctgtgcgtcaaggagggcatggccaagaacacctacggcaccggctgcttcatgctgatgaacaccggcgagaaggccgtcaagagcgagaacggcctgctgaccaccatcgcctgcggcccgaccggcgaggtcaactacgccctggagggcgccgtcttcatggccggcgccagcatccagtggctgcgcgacgagatgaagctgatcaacgacgcctacgacagcgagtacttcgccaccaaggtccagaacaccaacggcgtctacgtcgtcccggccttcaccggcctgggcgccccgtactgggacccgtacgcccgcggcgccatcttcggcctgacccgcggcgtcaacgccaaccacatcatccgcgccaccctggagagcatcgcctaccagacccgcgacgtcctggaggccatgcaggccgacagcggcatccgcctgcacgccctgcgcgtcgacggcggcgccgtcgccaacaacttcctgatgcagttccagagcgacatcctgggcacccgcgtcgagcgcccggaggtccgcgaggtcaccgccctgggcgccgcctacctggccggcctggccgtcggcttctggcagaacctggacgagctgcaggtgaaggccgtcatcgagcgcgagttccgcccgggcatcgagaccaccgagcgcaactaccgctacgccggctggaagaaggccgtcaagcgcgccatggcctgggaggagcacgacgagtga SEQ ID Intergenic TCATTCTTGGAGGAGACACNO: 98 region SEQ ID GlpDatggagaccaaggacctgatcgtcatcggcggcggcatcaacggcgccgg NO: 99catcgccgccgacgccgccggccgcggcctgagcgtcctgatgctggaggcccaggacctggcctgcgccaccagcagcgccagcagcaagctgatccacggcggcctgcgctacctggagcactacgagttccgcctggtcagcgaggccctggccgagcgcgaggtcctgctgaagatggccccgcacatcgccttcccgatgcgcttccgcctgccgcaccgcccgcacctgcgcccggcctggatgatccgcatcggcctgttcatgtacgaccacctgggcaagcgcaccagcctgccgggcagcaccggcctgcgcttcggcgccaacagcgtcctgaagccggagatcaagcgcggcttcgagtacagcgactgctgggtcgacgacgcccgcctggtcctggccaacgcccagatggtcgtccgcaagggcggcgaggtcctgacccgcacccgcgccaccagcgcccgccgcgagaacggcctgtggatcgtcgaggccgaggacatcgacaccggcaagaagtacagctggcaggcccgcggcctggtcaacgccaccggcccgtgggtcaagcagttcttcgacgacggcatgcacctgccgagcccgtacggcatccgcctgatcaagggcagccacatcgtcgtcccgcgcgtccacacccagaagcaggcctacatcctgcagaacgaggacaagcgcatcgtcttcgtcatcccgtggatggacgagttcagcatcatcggcaccaccgacgtcgagtacaagggcgacccgaaggccgtcaagatcgaggagagcgagatcaactacctgctgaacgtctacaacacccacttcaagaagcagctgagccgcgacgacatcgtctggacctacagcggcgtccgcccgctgtgcgacgacgagagcgacagcccgcaggccatcacccgcgactacaccctggacatccacgacgagaacggcaaggccccgctgctgagcgtcttcggcggcaagctgaccacctaccgcaagctggccgagcacgccctggagaagctgaccccgtactaccagggcatcggcccggcctggaccaaggagagcgtcctgccgggcggcgccatcgagggcgaccgcgacgactacgccgcccgcctgcgccgccgctacccgttcctgaccgagagcctggcccgccactacgcccgcacctacggcagcaacagcgagctgctgctgggcaacgccggcaccgtcagcgacctgggcgaggacttcggccacgagttctacgaggccgagctgaagtacctggtcgaccacgagtgggtccgccgcgccgacgacgccctgtggcgccgcaccaagcagggcatgtggctgaacgccgaccagcagagccgcgtcagccagtggctggtcgagtacacccagcagcgcctgagcctggccagctga SEQ ID Intergenic TCATTCTTGGAGGAGACACNO: 100 region SEQ ID GlpFatgagccagaccagcaccctgaagggccagtgcatcgccgagttcctgggc NO: 101accggcctgctgatcttcttcggcgtcggctgcgtcgccgccctgaaggtcgccggcgccagcttcggccagtgggagatcagcgtcatctggggcctgggcgtcgccatggccatctacctgaccgccggcgtcagcggcgcccacctgaacccggccgtcaccatcgccctgtggctgttcgcctgcttcgacaagcgcaaggtcatcccgttcatcgtcagccaggtcgccggcgccttctgcgccgccgccctggtctacggcctgtactacaacctgttcttcgacttcgagcagacccaccacatcgtccgcggcagcgtcgagagcgtcgacctggccggcaccttcagcacctacccgaacccgcacatcaacttcgtccaggccttcgccgtcgagatggtcatcaccgccatcctgatgggcctgatcctggccctgaccgacgacggcaacggcgtcccgcgcggcccgctggccccgctgctgatcggcctgctgatcgccgtcatcggcgccagcatgggcccgctgaccggcttcgccatgaacccggcccgcgacttcggcccgaaggtcttcgcctggctggccggctggggcaacgtcgccttcaccggcggccgcgacatcccgtacttcctggtcccgctgttcggcccgatcgtcggcgccatcgtcggcgccttcgcctaccgcaagctgatcggccgccacctgccgtgcgacatctgcgtcgtcgaggagaaggagaccaccaccccgagcgagc agaaggccagcctatgaSEQ ID Glycerol tttgcctcgatcggcggtccttgtgacagggagatattcccgacggatccgggNO: 102 Utilization gcattcgagcggaaccgcccgccgtgggagtttttccagcgagcattcgagaPathway gtttttcaaggcggcttcgaggggttattccgtaacgccgccgacatgatctgt Operoncccagaatctccgccgctgttcgtagagcgccgatgcagggtcggcatcaatcattcttggaggagacacatgaccgagaagaagtacatcgtcgccctggaccagggcaccaccagcagccgcgccgtcgtcatggaccacgacgccaacatcatcagcgtcagccagcgcgagttcgagcagatctacccgaagccgggctgggtcgagcacgacccgatggagatctgggccacccagagcagcaccctggtcgaggtcctggccaaggccgacatcagcagcgaccagatcgccgccatcggcatcaccaaccagcgcgagaccaccatcgtctgggagaaggagaccggcaagccgatctacaacgccatcgtctggcagtgccgccgcaccgccgagatctgcgagcacctgaagcgcgacggcctggaggactacatccgcagcaacaccggcctggtcatcgacccgtacttcagcggcaccaaggtcaagtggatcctggaccacgtcgagggcagccgcgagcgcgcccgccgcggcgagctgctgttcggcaccgtcgacacctggctgatctggaagatgacccagggccgcgtccacgtcaccgactacaccaacgccagccgcaccatgctgttcaacatccacaccctggactgggacgacaagatgctggaggtcctggacatcccgcgcgagatgctgccggaggtccgccgcagcagcgaggtctacggccagaccaacatcggcggcaagggcggcacccgcatcccgatcagcggcatcgccggcgaccagcaggccgccctgttcggccagctgtgcgtcaaggagggcatggccaagaacacctacggcaccggctgcttcatgctgatgaacaccggcgagaaggccgtcaagagcgagaacggcctgctgaccaccatcgcctgcggcccgaccggcgaggtcaactacgccctggagggcgccgtcttcatggccggcgccagcatccagtggctgcgcgacgagatgaagctgatcaacgacgcctacgacagcgagtacttcgccaccaaggtccagaacaccaacggcgtctacgtcgtcccggccttcaccggcctgggcgccccgtactgggacccgtacgcccgcggcgccatcttcggcctgacccgcggcgtcaacgccaaccacatcatccgcgccaccctggagagcatcgcctaccagacccgcgacgtcctggaggccatgcaggccgacagcggcatccgcctgcacgccctgcgcgtcgacggcggcgccgtcgccaacaacttcctgatgcagttccagagcgacatcctgggcacccgcgtcgagcgcccggaggtccgcgaggtcaccgccctgggcgccgcctacctggccggcctggccgtcggcttctggcagaacctggacgagctgcaggagaaggccgtcatcgagcgcgagttccgcccgggcatcgagaccaccgagcgcaactaccgctacgccggctggaagaaggccgtcaagcgcgccatggcctgggaggagcacgacgagtgatcattcttggaggagacacatggagaccaaggacctgatcgtcatcggcggcggcatcaacggcgccggcatcgccgccgacgccgccggccgcggcctgagcgtcctgatgctggaggcccaggacctggcctgcgccaccagcagcgccagcagcaagctgatccacggcggcctgcgctacctggagcactacgagttccgcctggtcagcgaggccctggccgagcgcgaggtcctgctgaagatggccccgcacatcgccttcccgatgcgcttccgcctgccgcaccgcccgcacctgcgcccggcctggatgatccgcatcggcctgttcatgtacgaccacctgggcaagcgcaccagcctgccgggcagcaccggcctgcgcttcggcgccaacagcgtcctgaagccggagatcaagcgcggcttcgagtacagcgactgctgggtcgacgacgcccgcctggtcctggccaacgcccagatggtcgtccgcaagggcggcgaggtcctgacccgcacccgcgccaccagcgcccgccgcgagaacggcctgtggatcgtcgaggccgaggacatcgacaccggcaagaagtacagctggcaggcccgcggcctggtcaacgccaccggcccgtgggtcaagcagttcttcgacgacggcatgcacctgccgagcccgtacggcatccgcctgatcaagggcagccacatcgtcgtcccgcgcgtccacacccagaagcaggcctacatcctgcagaacgaggacaagcgcatcgtcttcgtcatcccgtggatggacgagttcagcatcatcggcaccaccgacgtcgagtacaagggcgacccgaaggccgtcaagatcgaggagagcgagatcaactacctgctgaacgtctacaacacccacttcaagaagcagctgagccgcgacgacatcgtctggacctacagcggcgtccgcccgctgtgcgacgacgagagcgacagcccgcaggccatcacccgcgactacaccctggacatccacgacgagaacggcaaggccccgctgctgagcgtcttcggcggcaagctgaccacctaccgcaagctggccgagcacgccctggagaagctgaccccgtactaccagggcatcggcccggcctggaccaaggagagcgtcctgccgggcggcgccatcgagggcgaccgcgacgactacgccgcccgcctgcgccgccgctacccgttcctgaccgagagcctggcccgccactacgcccgcacctacggcagcaacagcgagctgctgctgggcaacgccggcaccgtcagcgacctgggcgaggacttcggccacgagttctacgaggccgagctgaagtacctggtcgaccacgagtgggtccgccgcgccgacgacgccctgtggcgccgcaccaagcagggcatgtggctgaacgccgaccagcagagccgcgtcagccagtggctggtcgagtacacccagcagcgcctgagcctggccagctgatcattcttggaggagacacatgagccagaccagcaccctgaagggccagtgcatcgccgagttcctgggcaccggcctgctgatcttcttcggcgtcggctgcgtcgccgccctgaaggtcgccggcgccagcttcggccagtgggagatcagcgtcatctggggcctgggcgtcgccatggccatctacctgaccgccggcgtcagcggcgcccacctgaacccggccgtcaccatcgccctgtggctgttcgcctgcttcgacaagcgcaaggtcatcccgttcatcgtcagccaggtcgccggcgccttctgcgccgccgccctggtctacggcctgtactacaacctgttcttcgacttcgagcagacccaccacatcgtccgcggcagcgtcgagagcgtcgacctggccggcaccttcagcacctacccgaacccgcacatcaacttcgtccaggccttcgccgtcgagatggtcatcaccgccatcctgatgggcctgatcctggccctgaccgacgacggcaacggcgtcccgcgcggcccgctggccccgctgctgatcggcctgctgatcgccgtcatcggcgccagcatgggcccgctgaccggcttcgccatgaacccggcccgcgacttcggcccgaaggtcttcgcctggctggccggctggggcaacgtcgccttcaccggcggccgcgacatcccgtacttcctggtcccgctgttcggcccgatcgtcggcgccatcgtcggcgccttcgcctaccgcaagctgatcggccgccacctgccgtgcgacatctgcgtcgtcgaggagaaggagaccaccaccccgagcgagcagaaggccagcctgtga

The synthetic operon is then cloned and transformed into M. capsulatusas described above. Transformation is confirmed by resistance of thecells to antibiotic selection, and gene expression is confirmed bynorthern blot (to confirm RNA transcription), western blot, or ELISAmethods (to confirm protein expression).

Growth on Glycerol as a Sole Carbon Source. Recombinant M. capsulatustransformed with a vector containing the synthetic operon encoding genesfor glycerol utilization are inoculated into 100 mL shake flaskscontaining 20-50 mL NMS media, 1% glycerol and 10 ug/mL kanamycin. Theflasks are then shaken continuously while being incubated at 42° C.Growth is confirmed by monitoring optical density of the culture overtime. Note that because glycerol is the only carbon source provided tothe cells, all cell mass produced must have been derived from glycerol.

Example 3 Recombinant Methylomonas Methanica Engineered to Grow onGlycerol

Growth and Conjugations. The procedure for growth and conjugation ofMethylomonas methanica was performed essentially identically to theprocedures described for M. capsulatus (above).

Introduction of Glycerol Utilization Pathway. Nucleic acid sequencesencoding GlpK, GlpD, and GlpF from E. coli were codon optimized forexpression in M. methanica. The codon optimized nucleic acids encodingGlpK, GlpD, and GlpF are synthesized as an operon (SEQ ID NO:109; seeTable 16 for components of operon) under control of an hps promoter withappropriate intergenic regions (CAPITALIZED sequence) incorporatingribosome binding sequences.

TABLE 16Glycerol Utilization Pathway Operon Codon Optimized for M. methanicaSEQ ID NO: # Gene Name Nucleotide Sequence SEQ ID HPSTTCGGAATCCCTGACGGGAATTGGCCCGA NO: 103 promoterAGAAGGCAGATGCCATCGTTCAGTATCGA AAGGAACATGGGGATTTTCAGTCATTGAAGGATCTGGAGAATGTCAGCGGCATTGGCG AGAAAACCCTTCAGGCCAATGAAAAAGACATTCGCTTCACGGATGATTTGAGCGATAAG TCATCCGCGGAAAAAGGTGCGGTAGCTGTGGATAAAAAAGGCGCCAGATAGTAAGCGC TAAGGATTGGGGTGCGTCGCCGGTCGCGGCGGCGCTCCTCGACGGCAGAGTTGGTGCC AGGTTGGCGGATGATTGATGCCGAATATTACGCGACCAATTCTCGAGGCAAATGAACT GTGAGCTACTGAGTTGCAGGCATTGACAGCCATCCCATTTCTATCATACAGTTACGGAC GCATCACGAGTAGGTGATAAGCCTAGCAGATTGCGGCAGTTGGCAAAATCAGCTATTAC TAATAATTAAAAACTTTCGGAGCACATCAC SEQ IDGlpK atgaccgaaaaaaaatatatcgtcgcgttggatcaaggcaccaccag NO: 104cagccgcgcggtcgtcatggatcacgatgcgaacatcatcagcgtcagccaacgcgaattcgaacaaatctatccgaaaccgggctgggtcgaacacgatccgatggaaatctgggcgacccaaagcagcaccttggtcgaagtcttggcgaaagcggatatcagcagcgatcaaatcgcggcgatcggcatcaccaaccaacgcgaaaccaccatcgtctgggaaaaagaaaccggcaaaccgatctataacgcgatcgtctggcaatgccgccgcaccgcggaaatctgcgaacacttgaaacgcgatggcttggaagattatatccgcagcaacaccggcttggtcatcgatccgtatttcagcggcaccaaagtcaaatggatcttggatcacgtcgaaggcagccgcgaacgcgcgcgccgcggcgaattgttgttcggcaccgtcgatacctggttgatctggaaaatgacccaaggccgcgtccacgtcaccgattataccaacgcgagccgcaccatgttgttcaacatccacaccttggattgggatgataaaatgttggaagtcttggatatcccgcgcgaaatgttgccggaagtccgccgcagcagcgaagtctatggccaaaccaacatcggcggcaaaggcggcacccgcatcccgatcagcggcatcgcgggcgatcaacaagcggcgttgttcggccaattgtgcgtcaaagaaggcatggcgaaaaacacctatggcaccggctgcttcatgttgatgaacaccggcgaaaaagcggtcaaaagcgaaaacggcttgttgaccaccatcgcgtgcggcccgaccggcgaagtcaactatgcgttggaaggcgcggtcttcatggcgggcgcgagcatccaatggttgcgcgatgaaatgaaattgatcaacgatgcgtatgatagcgaatatttcgcgaccaaagtccaaaacaccaacggcgtctatgtcgtcccggcgttcaccggcttgggcgcgccgtattgggatccgtatgcgcgcggcgcgatcttcggcttgacccgcggcgtcaacgcgaaccacatcatccgcgcgaccttggaaagcatcgcgtatcaaacccgcgatgtcttggaagcgatgcaagcggatagcggcatccgcttgcacgcgttgcgcgtcgatggcggcgcggtcgcgaacaacttcttgatgcaattccaaagcgatatcttgggcacccgcgtcgaacgcccggaagtccgcgaagtcaccgcgttgggcgcggcgtatttggcgggcttggcggtcggcttctggcaaaacttggatgaattgcaagaaaaagcggtcatcgaacgcgaattccgcccgggcatcgaaaccaccgaacgcaactatcgctatgcgggctggaaaaaagcggtcaaacgcgcgatggcgtgggaagaacacgatg aataa SEQ ID IntergenicTAATAATTAAAAACTTTCGGAGCACATCAC NO: 105 region SEQ ID GlpDatggaaaccaaagatttgatcgtcatcggcggcggcatcaacggcgc NO: 106gggcatcgcggcggatgcggcgggccgcggcttgagcgtcttgatgttggaagcgcaagatttggcgtgcgcgaccagcagcgcgagcagcaaattgatccacggcggcttgcgctatttggaacactatgaattccgcttggtcagcgaagcgttggcggaacgcgaagtcttgttgaaaatggcgccgcacatcgcgttcccgatgcgcttccgcttgccgcaccgcccgcacttgcgcccggcgtggatgatccgcatcggcttgttcatgtatgatcacttgggcaaacgcaccagcttgccgggcagcaccggcttgcgcttcggcgcgaacagcgtcttgaaaccggaaatcaaacgcggcttcgaatatagcgattgctgggtcgatgatgcgcgcttggtcttggcgaacgcgcaaatggtcgtccgcaaaggcggcgaagtcttgacccgcacccgcgcgaccagcgcgcgccgcgaaaacggcttgtggatcgtcgaagcggaagatatcgataccggcaaaaaatatagctggcaagcgcgcggcttggtcaacgcgaccggcccgtgggtcaaacaattcttcgatgatggcatgcacttgccgagcccgtatggcatccgcttgatcaaaggcagccacatcgtcgtcccgcgcgtccacacccaaaaacaagcgtatatcttgcaaaacgaagataaacgcatcgtcttcgtcatcccgtggatggatgaattcagcatcatcggcaccaccgatgtcgaatataaaggcgatccgaaagcggtcaaaatcgaagaaagcgaaatcaactatttgttgaacgtctataacacccacttcaaaaaacaattgagccgcgatgatatcgtctggacctatagcggcgtccgcccgttgtgcgatgatgaaagcgatagcccgcaagcgatcacccgcgattataccttggatatccacgatgaaaacggcaaagcgccgttgttgagcgtcttcggcggcaaattgaccacctatcgcaaattggcggaacacgcgttggaaaaattgaccccgtattatcaaggcatcggcccggcgtggaccaaagaaagcgtcttgccgggcggcgcgatcgaaggcgatcgcgatgartatgcggcgcgcttgcgccgccgctatccgttcttgaccgaaagcttggcgcgccactatgcgcgcacctatggcagcaacagcgaattgttgttgggcaacgcgggcaccgtcagcgatttgggcgaagamcggccacgaattctatgaagcggaattgaaatatttggtcgatcacgaatgggtccgccgcgcggatgatgcgttgtggcgccgcaccaaacaaggcatgtggttgaacgcggatcaacaaagccgcgtcagccaatggttggtcgaatatacccaacaacgcttgagcttggcgagctaa SEQ ID IntergenicTAATAATTAAAAACTTTCGGAGCACATCAC NO: 107 region SEQ ID GlpFatgagccaaaccagcaccttgaaaggccaatgcatcgcggaattcttg NO: 108ggcaccggcttgttgatcttcttcggcgtcggctgcgtcgcggcgttgaaagtcgcgggcgcgagcttcggccaatgggaaatcagcgtcatctggggcttgggcgtcgcgatggcgatctatttgaccgcgggcgtcagcggcgcgcacttgaacccggcggtcaccatcgcgttgtggttgttcgcgtgcttcgataaacgcaaagtcatcccgttcatcgtcagccaagtcgcgggcgcgttctgcgcggcggcgttggtctatggcttgtattataacttgttcttcgatttcgaacaaacccaccacatcgtccgcggcagcgtcgaaagcgtcgatttggcgggcaccttcagcacctatccgaacccgcacatcaacttcgtccaagcgttcgcggtcgaaatggtcatcaccgcgatcttgatgggcttgatcttggcgttgaccgatgatggcaacggcgtcccgcgcggcccgttggcgccgttgttgatcggcttgttgatcgcggtcatcggcgcgagcatgggcccgttgaccggcttcgcgatgaacccggcgcgcgatttcggcccgaaagtcttcgcgtggttggcgggctggggcaacgtcgcgttcaccggcggccgcgatatcccgtatttcttggtcccgttgttcggcccgatcgtcggcgcgatcgtcggcgcgttcgcgtatcgcaaattgatcggccgccacttgccgtgcgatatctgcgtcgtcgaagaaaaagaaaccaccaccccgagcgaacaaaaagcgagcttgtaa SEQ ID GlycerolTTCGGAATCCCTGACGGGAATTGGCCCGA NO: 109 UtilizationAGAAGGCAGATGCCATCGTTCAGTATCGA Pathway AAGGAACATGGGGATTTTCAGTCATTGAAOperon GGATCTGGAGAATGTCAGCGGCATTGGCG AGAAAACCCTTCAGGCCAATGAAAAAGACATTCGCTTCACGGATGATTTGAGCGATAAG TCATCCGCGGAAAAAGGTGCGGTAGCTGTGGATAAAAAAGGCGCCAGATAGTAAGCGC TAAGGATTGGGGTGCGTCGCCGGTCGCGGCGGCGCTCCTCGACGGCAGAGTTGGTGCC AGGTTGGCGGATGATTGATGCCGAATATTACGCGACCAATTCTCGAGGCAAATGAACT GTGAGCTACTGAGTTGCAGGCATTGACAGCCATCCCATTTCTATCATACAGTTACGGAC GCATCACGAGTAGGTGATAAGCCTAGCAGATTGCGGCAGTTGGCAAAATCAGCTATTAC TAATAATTAAAAACTTTCGGAGCACATCACatgaccgaaaaaaaatatatcgtcgcgttggatcaaggcaccaccagcagccgcgcggtcgtcatggatcacgatgcgaacatcatcagcgtcagccaacgcgaattcgaacaaatctatccgaaaccgggctgggtcgaacacgatccgatggaaatctgggcgacccaaagcagcaccttggtcgaagtcttggcgaaagcggatatcagcagcgatcaaatcgcggcgatcggcatcaccaaccaacgcgaaaccaccatcgtctgggaaaaagaaaccggcaaaccgatctataacgcgatcgtctggcaatgccgccgcaccgcggaaatctgcgaacacttgaaacgcgatggcttggaagattatatccgcagcaacaccggcttggtcatcgatccgtatttcagcggcaccaaagtcaaatggatcttggatcacgtcgaaggcagccgcgaacgcgcgcgccgcggcgaattgttgttcggcaccgtcgatacctggttgatctggaaaatgacccaaggccgcgtccacgtcaccgattataccaacgcgagccgcaccatgttgttcaacatccacaccttggattgggatgataaaatgttggaagtcttggatatcccgcgcgaaatgttgccggaagtccgccgcagcagcgaagtctatggccaaaccaacateggcggcaaaggcggcacccgcatcccgatcagcggcatcgcgggcgatcaacaagcggcgttgttcggccaattgtgcgtcaaagaaggcatggcgaaaaacacctatggcaccggctgcttcatgttgatgaacaccggcgaaaaagcggtcaaaagcgaaaacggcttgttgaccaccatcgcgtgcggcccgaccggcgaagtcaactatgcgttggaaggcgcggtcttcatggcgggcgcgagcatccaatggttgcgcgatgaaatgaaattgatcaacgatgcgtatgatagcgaatatttcgcgaccaaagtccaaaacaccaacggcgtctatgtcgtcccggcgttcaccggcttgggcgcgccgtattgggatccgtatgcgcgcggcgcgatcttcggcttgacccgcggcgtcaacgcgaaccacatcatccgcgcgaccttggaaagcatcgcgtatcaaacccgcgatgtcttggaagcgatgcaagcggatagcggcatccgcttgcacgcgttgcgcgtcgatggcggcgcggtcgcgaacaacttcttgatgcaattccaaagcgatatcttgggcacccgcgtcgaacgcccggaagtccgcgaagtcaccgcgttgggcgcggcgtatttggcgggcttggcggtcggcttctggcaaaacttggatgaattgcaagaaaaagcggtcatcgaacgcgaattccgcccgggcatcgaaaccaccgaacgcaactatcgctatgcgggctggaaaaaagcggtcaaacgcgcgatggcgtgggaagaacacgatgaataaTAATAATTAAAAACTTTCGGAGCACATCACatggaaaccaaagatttgatcgtcatcggcggcggcatcaacggcgcgggcatcgcggcggatgcggcgggccgcggcttgagcgtcttgatgttggaagcgcaagatttggcgtgcgcgaccagcagcgcgagcagcaaattgatccacggcggcttgcgctatttggaacactatgaattccgcttggtcagcgaagcgttggcggaacgcgaagtcttgttgaaaatggcgccgcacatcgcgttcccgatgcgcttccgcttgccgcaccgcccgcacttgcgcccggcgtggatgatccgcatcggcttgttcatgtatgatcacttgggcaaacgcaccagcttgccgggcagcaccggcttgcgcttcggcgcgaacagcgtcttgaaaccggaaatcaaacgcggcttcgaatatagcgattgctgggtcgatgatgcgcgcttggtcttggcgaacgcgcaaatggtcgtccgcaaaggcggcgaagtcttgacccgcacccgcgcgaccagcgcgcgccgcgaaaacggcttgtggatcgtcgaagcggaagatatcyataccggcaaaaaatatagctggcaagcgcgcggcttggtcaacgcgaccggcccgtgggtcaaacaattcttcgatgatggcatgcacttgccgagcccgtatggcatccgcttgatcaaaggcagccacatcgtcgtcccgcgcgtccacacccaaaaacaagcgtatatcttgcaaaacgaagataaacgcatcgtcttcgtcatcccgtggatggatgaattcagcatcatcggcaccaccgatgtcgaatataaaggcgatccgaaagcggtcaaaatcgaagaaagcgaaatcaactatttgttgaacgtctataacacccacttcaaaaaacaattgagccgcgatgatatcgtctggacctatagcggcgtccgcccgttgtgcgatgatgaaagcgatagcccgcaagcgatcacccgcgattataccttggatatccacgatgaaaacggcaaagcgccgttgttgagcgtcttcggcggcaaattgaccacctatcgcaaattggcggaacacgcgttggaaaaattgaccccgtattatcaaggcatcggcccggcgtggaccaaagaaagcgtcttgccgggcggcgcgatcgaaggcgatcgcgatgattatgcggcgcgcttgcgccgccgctatccgttcttgaccgaaagcttggcgcgccactatgcgcgcacctatggcagcaacagcgaattgttgttgggcaacgcgggcaccgtcagcgatttgggcgaagatttcggccacgaattctatgaagcggaattgaaatatttggtcgatcacgaatgggtccgccgcgcggatgatgcgttgtggcgccgcaccaaacaaggcatgtggttgaacgcggatcaacaaagccgcgtcagccaatggttggtcgaatatacccaacaacgcttgagcttggcgagctaaTAATAATTAAAAACTTTCGGAGCACATCACatgagccaaaccagcaccttgaaaggccaatgcatcgcggaattcttgggcaccggcttgttgatcttcttcggcgtcggctgcgtcgcggcgttgaaagtcgcgggcgcgagcttcggccaatgggaaatcagcgtcatctggggcttgggcgtcgcgatggcgatctatttgaccgcgggcgtcagcggcgcgcacttgaacccggcggtcaccatcgcgttgtggttgttcgcgtgcttcgataaacgcaaagtcatcccgttcatcgtcagccaagtcgcgggcgcgttctgcgcggcggcgttggtctatggcttgtattataacttgttcttcgatttcgaacaaacccaccacatcgtccgcggcagcgtcgaaagcgtcgatttggcgggcaccttcagcacctatccgaacccgcacatcaacttcgtccaagcgttcgcggtcgaaatggtcatcaccgcgatcttgatgggcttgatcttggcgttgaccgatgatggcaacggcgtcccgcgcggcccgttggcgccgttgttgatcggcttgttgatcgcggtcatcggcgcgagcatgggcccgttgaccggcttcgcgatgaacccggcgcgcgatttcggcccgaaagtcttcgcgtggttggcgggctggggcaacgtcgcgttcaccggcggccgcgatatcccgtatttcttggtcccgttgttcggcccgatcgtcggcgcgatcgtcggcgcgttcgcgtatcgcaaattgatcggccgccacttgccgtgcgatatctgcgtcgtcgaagaaaaagaaaccaccaccccgagcgaacaaaaagcgagcttgtaa

The synthetic operon is then cloned and transformed into M. methanica asdescribed above. Transformation is confirmed by resistance of the cellsto antibiotic selection, and gene expression is confirmed by northernblot (to confirm RNA transcription), western blot, or ELISA methods (toconfirm protein expression).

Growth on Glycerol as a Sole Carbon Source. Recombinant M. methanicatransformed with a vector containing the synthetic operon encoding genesfor glycerol utilization are inoculated into 100 mL shake flaskscontaining 20-50 mL NMS media, 1% glycerol and 10 ug/mL kanamycin. Theflasks are then shaken continuously while being incubated at 30° C.Growth is confirmed by monitoring optical density of the culture overtime. Note that because glycerol is the only carbon source provided tothe cells, all cell mass produced must have been derived from glycerol.

Example 4 Recombinant Methylosinus Trichosporium Engineered to Grow onAcetate

M. trichosporium cells are cultured and conjugated as described above.

Introduction of an Acetate Utilization Pathway. Nucleic acid sequencesencoding AcsA (acetyl-CoA synthase) and ActP from E. coli were codonoptimized for expression in M. trichosporium. The codon optimizednucleic acids encoding AcsA and ActP are synthesized as an operon (SEQID NO:114; see Table 17 for components of operon) under control of anmdh promoter with appropriate intergenic regions (CAPITALIZED sequence)incorporating ribosome binding sequences.

TABLE 17Acetate Utilization Pathway Operon Codon Optimized for M. trichosporiumSEQ ID NO: # Gene Name Nucleotide Sequence SEQ ID MDHTTTGCCTCGATCGGCGGTCCTTGTGACAGGGA NO: 110 promoterGATATTCCCGACGGATCCGGGGCATTCGAGCG GAACCGCCCGCCGTGGGAGTTTTTCCAGCGAGCATTCGAGAGTTTTTCAAGGCGGCTTCGAGGG GTTATTCCGTAACGCCGCCGACATGATCTGTCCCAGAATCTCCGCCGCTGTTCGTAGAGCGCCGA TGCAGGGTCGGCATCAATCATTCTTGGAGGAG ACACSEQ ID AcsA atgtcagatccataagcataccatcccggcgaacatcgcggaccgctgc NO: 111ctgatcaacccgcagcagtatgaggcgatgtatcagcagtcgatcaacgtcccggacaccttctggggcgagcagggcaagatcctggactggatcaagccgtatcagaaggtcaagaacacctcgttcgcgccgggcaacgtctcgatcaagtggtatgaggacggcaccctgaacctggcggcgaactgcctggaccgccatctgcaggagaacggcgaccgcaccgcgatcatctgggagggcgacgacgcgtcgcagtcgaagcatatctcgtataaggagctgcatcgcgacgtctgccgcttcgcgaacaccctgctggagctgggcatcaagaagggcgacgtcgtcgcgatctatatgccgatggtcccggaggcggcggtcgcgatgctggcgtgcgcgcgcatcggcgcggtccattcggtcatcttcggcggcttctcgccggaggcggtcgcgggccgcatcatcgactcgaactcgcgcctggtcatcacctcggacgagggcgtccgcgcgggccgctcgatcccgctgaagaagaacgtcgacgacgcgctgaagaacccgaacgtcacctcggtcgagcatgtcgtcgtcctgaagcgcaccggcggcaagatcgactggcaggagggccgcgacctgtggtggcatgacctggtcgagcaggcgtcggaccagcatcaggcggaggagatgaacgcggaggacccgctgttcatcctgtatacctcgggctcgaccggcaagccgaagggcgtcctgcataccaccggcggctatctggtctatgcggcgctgaccttcaagtatgtcttcgactatcatccgggcgacatctattggtgcaccgcggacgtcggctgggtcaccggccattcgtatctgctgtatggcccgctggcgtgcggcgcgaccaccctgatgttcgagggcgtcccgaactggccgaccccggcgcgcatggcgcaggtcgtcgacaagcatcaggtcaacatcctgtataccgcgccgaccgcgatccgcgcgctgatggcggagggcgacaaggcgatcgagggcaccgaccgctcgtcgctgcgcatcctgggctcggtcggcgagccgatcaacccggaggcgtgggagtggtattggaagaagatcggcaacgagaagtgcccggtcgtcgacacctggtggcagaccgagaccggcggcttcatgatcaccccgctgccgggcgcgaccgagctgaaggcgggctcggcgacccgcccgttcttcggcgtccagccggcgctggtcgacaacgagggcaacccgctggagggcgcgaccgagggctcgctggtcatcaccgactcgtggccgggccaggcgcgcaccctgttcggcgaccatgagcgcttcgagcagacctatttctcgaccttcaagaacatgtatttctcgggcgacggcgcgcgccgcgacgaggacggctattattggatcaccggccgcgtcgacgacgtcctgaacgtctcgggccatcgcctgggctccgcggagatcgagtcggcgctggtcgcgcatccgaagatcgcggaggcggcggtcgtcggcatcccgcataacatcaagggccaggcgatctatgcgtatgtcaccctgaaccatggcgaggagccgtcgccggagctgtatgcggaggtccgcaactgggtccgcaaggagatcggcccgctggcgaccccggacgtcctgcattggaccgactcgctgccgaagacccgctcgggcaagatcatgcgccgcatcctgcgcaagatcgcggcgggcgacacctcgaacctgggcgacacctcgaccctggcggacccgggcgtcgtcgagaagccgctggaggagaagcaggcgatcgcgatgccgtcgtga SEQ ID Intergenic TCATTCTTGGAGGAGACACNO: 112 Region SEQ ID ActPatgaagcgcgtcctgaccgcgctggcggcgaccctgccgttcgcggcgaa NO: 113cgcggcggacgcgatctcgggcgcggtcgagcgccagccgaccaactggcaggcgatcatcatgttcctgatcttcgtcgtcttcaccctgggcatcacctattgggcgtcgaagcgcgtccgctcgcgctcggactattataccgcgggcggcaacatcaccggcttccagaacggcctggcgatcgcgggcgactatatgtcggcggcgtcgttcctgggcatctcggcgctggtcttcacctcgggctatgacggcctgatctattcgctgggcttcctggtcggctggccgatcatcctgttcctgatcgcggagcgcctgcgcaacctgggccgctataccttcgcggacgtcgcgtcgtatcgcctgaagcagggcccgatccgcatcctgtcggcgtgcggctcgctggtcgtcgtcgcgctgtatctgatcgcgcagatggtcggcgcgggcaagctgatcgagctgctgttcggcctgaactatcatatcgcggtcgtcctggtcggcgtcctgatgatgatgtatgtcctgttcggcggcatgctggcgaccacctgggtccagatcatcaaggcggtcctgctgctgttcggcgcgtcgttcatggcgttcatggtcatgaagcatgtcggcttctcgttcaacaacctgttctcggaggcgatggcggtccatccgaagggcgtcgacatcatgaagccgggcggcctggtcaaggacccgatctcggcgctgtcgctgggcctgggcctgatgttcggcaccgcgggcctgccgcatatcctgatgcgcttcttcaccgtctcggacgcgcgcgaggcgcgcaagtcggtcttctatgcgaccggcttcatgggctatttctatatcctgaccttcatcatcggcttcggcgcgatcatgctggtcggcgcgaacccggagtataaggacgcggcgggccatctgatcggcggcaacaacatggcggcggtccatctggcgaacgcggtcggcggcaacctgttcctgggcttcatctcggcggtcgcgttcgcgaccatcctggcggtcgtcgcgggcctgaccctggcgggcgcgtcggcggtctcgcatgacctgtatgcgaacgtcttcaagaagggcgcgaccgagcgcgaggagctgcgcgtctcgaagatcaccgtcctgatcctgggcgtcatcgcgatcatcctgggcgtcctgttcgagaaccagaacatcgcgttcatggtcggcctggcgttcgcgatcgcggcgtcgtgcaacttcccgatcatcctgctgtcgatgtattggtcgaagctgaccacccgcggcgcgatgatgggcggctggctgggcctgatcaccgcggtcgtcctgatgatcctgggcccgaccatctgggtccagatcctgggccatgagaaggcgatcttcccgtatgagtatccggcgctgttctcgatcaccgtcgcgttcctgggcatctggttcttctcggcgaccgacaactcggcggagggcgcgcgcgagcgcgagctgttccgcgcgcagttcatccgctcgcagaccggcttcggcgtcgagcagggccgcgcgcattga SEQ ID AcetateTTTGCCTCGATCGGCGGTCCTTGTGACAGGGA NO: 114 UtilizationGATATTCCCGACGGATCCGGGGCATTCGAGCG PathwayGAACCGCCCGCCGTGGGAGTTTTTCCAGCGAG Operon CATTCGAGAGTTTTTCAAGGCGGCTTCGAGGGGTTATTCCGTAACGCCGCCGACATGATCTGTCC CAGAATCTCCGCCGCTGTTCGTAGAGCGCCGATGCAGGGTCGGCATCAATCATTCTTGGAGGAGACACatgtcgcagatccataagcataccatcccggcgaacatcgcggaccgctgcctgatcaacccgcagcagtatgaggcgatgtatcagcagtcgatcaacgtcccggacaccttctggggcgagcagggcaagatcctggactggatcaagccgtatcagaaggtcaagaacacctcgttcgcgccgggcaacgtctcgatcaagtggtatgaggacggcaccctgaacctggcggcgaactgcctggaccgccatctgcaggagaacggcgaccgcaccgcgatcatctgggagggcgacgacgcgtcgcagtcgaagcatatctcgtataaggagctgcatcgcgacgtctgccgcttcgcgaacaccctgctggagctgggcatcaagaagggcgacgtcgtcgcgatctatatgccgatggtcccggaggcggcggtcgcgatgctggcgtgcgcgcgcatcggcgcggtccattcggtcatcttcggcggcttctcgccggaggcggtcgcgggccgcatcatcgactcgaactcgcgcctggtcatcacctcggacgagggcgtccgcgcgggccgctcgatcccgctgaagaagaacgtcgacgacgcgctgaagaacccgaacgtcacctcggtcgagcatgtcgtcgtcctgaagcgcaccggcggcaagatcgactggcaggagggccgcgacctgtggtggcatgacctggtcgagcaggcgtcggaccagcatcaggcggaggagatgaacgcggaggacccgctgttcatcctgtatacctcgggctcgaccggcaagccgaagggcgtcctgcataccaccggcggctatctggtctatgcggcgctgaccttcaagtatgtcttcgactatcatccgggcgacatctattggtgcaccgcggacgtcggctgggtcaccggccattcgtatctgctgtatggcccgctggcgtgcggcgcgaccaccctgatgttcgagggcgtcccgaactggccgaccccggcgcgcatggcgcaggtcgtcgacaagcatcaggtcaacatcctgtataccgcgccgaccgcgatccgcgcgctgatggcggagggcgacaaggcgatcgagggcaccgaccgctcgtcgctgcgcatcctgggctcggtcggcgagccgatcaacccggaggcgtgggagtggtattggaagaagatcggcaacgagaagtgcccggtcgtcgacacctggtggcagaccgagaccggcggcttcatgatcaccccgctgccgggcgcgaccgagctgaaggcgggctcggcgacccgcccgttcttcggcgtccagccggcgctggtcgacaacgagggcaacccgctggagggcgcgaccgagggctcgctggtcatcaccgactcgtggccgggccaggcgcgcaccctgttcggcgaccatgagcgcttcgagcagacctatttctcgacctcaagaacatgtatttctcgggcgacggcgcgcgccgcgacgaggacggctattattggatcaccggccgcgtcgacgacgtcctgaacgtctcgggccatcgcctgggcaccgcggagatcgagtcggcgctggtcgcgcatccgaagatcgcggaggcggcggtcgtcggcatcccgcataacatcaagggccaggcgatctatgcgtatgtcaccctgaaccatggcgaggagccgtcgccggagctgtatgcggaggtccgcaactgggtccgcaaggagatcggcccgctggcgaccccggacgtcctgcattggaccgactcgctgccgaagacccgctcgggcaagatcatgcgccgcatcctgcgcaagatcgcggcgggcgacacctcgaacctgggcgacacctcgaccctggcggacccgggcgtcgtcgagaagctgctggaggagaagcaggcgatcgcgatgccgtcgtgaTCATTCTTGGAGGAGACACatgaagcgcgtcctgaccgcgctggcggcgaccctgccgttcgcggcgaacgcggcggacgcgatctcgggcgcggtcgagcgccagccgaccaactggcaggcgatcatcatgttcctgatcttcgtcgtcttcaccctgggcatcacctattgggcgtcgaagcgcgtccgctcgcgctcggactattataccgcgggcggcaacatcaccggcttccagaacggcctggcgatcgcgggcgactatatgtcggcggcgtcgttcctgggcatctcggcgctggtcttcacctcgggctatgacggcctgatctattcgctgggcttcctggtcggctggccgatcatcctgttcctgatcgcggagcgcctgcgcaacctgggccgctataccttcgcggacgtcgcgtcgtatcgcctgaagcagggcccgatccgcatcctgtcggcgtgcggctcgctggtcgtcgtcgcgctgtatctgatcgcgcagatggtcggcgcgggcaagctgatcgagctgctgttcggcctgaactatcatatcgcggtcgtcctggtcggcgtcctgatgatgatgtatgtcctgttcggcggcatgctggcgaccacctgggtccagatcatcaaggcggtcctgctgctgttcggcgcgtcgttcatggcgttcatggtcatgaagcatgtcggcttctcgttcaacaacctgttctcggaggcgatggcggtccatccgaagggcgtcgacatcatgaagccgggcggcctggtcaaggacccgatctcggcgctgtcgctgggcctgggcctgatgttcggcaccgcgggcctgccgcatatcctgatgcgcttcttcaccgtctcggacgcgcgcgaggcgcgcaagtcggtcttctatgcgaccggcttcatgggctatttctatatcctgaccttcatcatcggcttcggcgcgatcatgctggtcggcgcgaacccggagtataaggacgcggcgggccatctgatcggcggcaacaacatggcggcggtccatctggcgaacgcggtcggcggcaacctgttcctgggcttcatctcggcggtcgcgttcgcgaccatcctggcggtcgtcgcgggcctgaccctggcgggcgcgtcggcggtctcgcatgacctgtatgcgaacgtcttcaagaagggcgcgaccgagcgcgaggagctgcgcgtctcgaagatcaccgtcctgatcctgggcgtcatcgcgatcatcctgggcgtcctgttcgagaaccagaacatcgcgttcatggtcggcctggcgttcgcgatcgcggcgtcgtgcaacttcccgatcatcctgctgtcgatgtattggtcgaagctgaccacccgcggcgcgatgatgggcggctggctgggcctgatcaccgcggtcgtcctgatgatcctgggcccgaccatctgggtccagatcctgggccatgagaaggcgatcttcccgtatgagtatccggcgctgttctcgatcaccgtcgcgttcctgggcatctggttcttctcggcgaccgacaactcggcggagggcgcgcgcgagcgcgagctgttccgcgcgcagttcatccgctcgcagaccggcttcggcgtcgagcagggccg cgcgcattga

The synthetic operon is then cloned and transformed as described above.Transformation is confirmed by resistance of the cells to antibioticselection, and gene expression is confirmed by northern blot (to confirmRNA transcription), western blot, or ELISA methods (to confirm proteinexpression).

Growth on Acetate as a Sole Carbon Source. Recombinant M. trichosporiumtransformed with a vector containing the synthetic operon encoding genesfor acetate utilization are inoculated into 100 mL shake flaskscontaining 20-50 mL NMS media, 1% sodium acetate and 10 ug/mL kanamycin.The flasks are then shaken continuously while being incubated at 30° C.Growth is confirmed by monitoring optical density of the culture overtime. Note that because acetate is the only carbon source provided tothe cells, all cell mass produced must have been derived from acetate.

Example 5 Recombinant Methylococcus Capsulatus Bath Engineered to Growon Acetate

M. capsulatus cells are cultured and conjugated as described above.

Introduction of an Acetate Utilization Pathway. Nucleic acid sequencesencoding AcsA (acetyl-CoA synthase) and ActP from E. coli were codonoptimized for expression in M. trichosporium. The codon optimizednucleic acids encoding AcsA and ActP are synthesized as an operon (SEQID NO:119; see Table 18 for components of operon) under control of anmdh promoter with appropriate intergenic regions (CAPITALIZED sequence)incorporating ribosome binding sequences.

TABLE 18Acetate Utilization Pathway Operon Codon Optimized for M. capsulatusSEQ ID NO: # Gene Name Nucleotide Sequence SEQ ID MDHTTTGCCTCGATCGGCGGTCCTTGTGACAGGG NO: 115 promoterAGATATTCCCGACGGATCCGGGGCATTCGAG CGGAACCGCCCGCCGTGGGAGTTTTTCCAGCGAGCATTCGAGAGTTTTTCAAGGCGGCTTCG AGGGGTTATTCCGTAACGCCGCCGACATGATCTGTCCCAGAATCTCCGCCGCTGTTCGTAGA GCGCCGATGCAGGGTCGGCATCAATCATTCTTGGAGGAGACAC SEQ ID AcsAatgagccagatccacaagcacaccatcccggccaacatcgccgaccgct NO: 116gcctgatcaacccgcagcagtacgaggccatgtaccagcagagcatcaacgtcccggacaccttctggggcgagcagggcaagatcctggactggatcaagccgtaccagaaggtcaagaacaccagcttcgccccgggcaacgtcagcatcaagtggtacgaggacggcaccctgaacctggccgccaactgcctggaccgccacctgcaggagaacggcgaccgcaccgccatcatctgggagggcgacgacgccagccagagcaagcacatcagctacaaggagctgcaccgcgacgtctgccgcttcgccaacaccctgctggagctgggcatcaagaagggcgacgtcgtcgccatctacatgccgatggtcccggaggccgccgtcgccatgctggcctgcgcccgcatcggcgccgtccacagcgtcatcttcggcggcttcagcccggaggccgtcgccggccgcatcatcgacagcaacagccgcctggtcatcaccagcgacgagggcgtccgcgccggccgcagcatcccgctgaagaagaacgtcgacgacgccctgaagaacccgaacgtcaccagcgtcgagcacgtcgtcgtcctgaagcgcaccggcggcaagatcgactggcaggagggccgcgacctgtggtggcacgacctggtcgagcaggccagcgaccagcaccaggccgaggagatgaacgccgaggacccgctgttcatcctgtacaccagcggcagcaccggcaagccgaagggcgtcctgcacaccaccggcggctacctggtctacgccgccctgaccttcaagtacgtcttcgactaccacccgggcgacatctactggtgcaccgccgacgtcggctgggtcaccggccacagctacctgctgtacggcccgctggcctgcggcgccaccaccctgatgttcgagggcgtcccgaactggccgaccccggcccgcatggcccaggtcgtcgacaagcaccaggtcaacatcctgtacaccgccccgaccgccatccgcgccctgatggccgagggcgacaaggccatcgagggcaccgaccgcagcagcctgcgcatcctgggcagcgtcggcgagccgatcaacccggaggcctgggagtggtactggaagaagatcggcaacgagaagtgcccggtcgtcgacacctggtggcagaccgagaccggcggcttcatgatcaccccgctgccgggcgccaccgagctgaaggccggcagcgccacccgcccgttcttcggcgtccagccggccctggtcgacaacgagggcaacccgctggagggcgccaccgagggcagcctggtcatcaccgacagctggccgggccaggcccgcaccctgttcggcgaccacgagcgcttcgagcagacctacttcagcaccttcaagaacatgtacttcagcggcgacggcgcccgccgcgacgaggacggctactactggatcaccggccgcgtcgacgacgtcctgaacgtcagcggccaccgcctgggcaccgccgagatcgagagcgccctggtcgcccacccgaagatcgccgaggccgccgtcgtcggcatcccgcacaacatcaagggccaggccatctacgcctacgtcaccctgaaccacggcgaggagccgagcccggagctgtacgccgaggtccgcaactgggtccgcaaggagatcggcccgctggccaccccggacgtcctgcactggaccgacagcctgccgaagacccgcagcggcaagatcatgcgccgcatcctgcgcaagatcgccgccggcgacaccagcaacctgggcgacaccagcaccctggccgacccgggcgtcgtcgagaagctgctggaggagaagcaggccatcgccatgccgagctga SEQ ID Intergenic TCATTCTTGGAGGAGACACNO: 117 Region SEQ ID ActPatgaagcgcgtcctgaccgccctggccgccaccctgccgttcgccgcca NO: 118acgccgccgacgccatcagcggcgccgtcgagcgccagccgaccaactggcaggccatcatcatgttcctgatcttcgtcgtcttcaccctgggcatcacctactgggccagcaagcgcgtccgcagccgcagcgactactacaccgccggcggcaacatcaccggcttccagaacggcctggccatcgccggcgactacatgagcgccgccagcttcctgggcatcagcgccctggtcttcaccagcggctacgacggcctgatctacagcctgggcttcctggtcggctggccgatcatcctgttcctgatcgccgagcgcctgcgcaacctgggccgctacaccttcgccgacgtcgccagctaccgcctgaagcagggcccgatccgcatcctgagcgcctgcggcagcctggtcgtcgtcgccctgtacctgatcgcccagatggtcggcgccggcaagctgatcgagctgctgttcggcctgaactaccacatcgccgtcgtcctggtcggcgtcctgatgatgatgtacgtcctgttcggcggcatgctggccaccacctgggtccagatcatcaaggccgtcctgctgctgttcggcgccagcttcatggccttcatggtcatgaagcacgtcggcttcagcttcaacaacctgttcagcgaggccatggccgtccacccgaagggcgtcgacatcatgaagccgggcggcctggtcaaggacccgatcagcgccctgagcctgggcctgggcctgatgttcggcaccgccggcctgccgcacatcctgatgcgcttcttcaccgtcagcgacgcccgcgaggcccgcaagagcgtcttctacgccaccggcttcatgggctacttctacatcctgaccttcatcatcggcttcggcgccatcatgctggtcggcgccaacccggagtacaaggacgccgccggccacctgatcggcggcaacaacatggccgccgtccacctggccaacgccgtcggcggcaacctgttcctgggcttcatcagcgccgtcgccttcgccaccatcctggccgtcgtcgccggcctgaccctggccggcgccagcgccgtcagccacgacctgtacgccaacgtcttcaagaagggcgccaccgagcgcgaggagctgcgcgtcagcaagatcaccgtcctgatcctgggcgtcatcgccatcatcctgggcgtcctgttcgagaaccagaacatcgccttcatggtcggcctggccttcgccatcgccgccagctgcaacttcccgatcatcctgctgagcatgtactggagcaagctgaccacccgcggcgccatgatgggcggctggctgggcctgatcaccgccgtcgtcctgatgatcctgggcccgaccatctgggtccagatcctgggccacgagaaggccatcttcccgtacgagtacccggccctgttcagcatcaccgtcgccttcctgggcatctggttcttcagcgccaccgacaacagcgccgagggcgcccgcgagcgcgagctgttccgcgcccagttcatccgcagccagaccggcttcggcgtcgag cagggccgcgcccactgaSEQ ID Acetate TTTGCCTCGATCGGCGGTCCTTGTGACAGGG NO: 119 UtilizationAGATATTCCCGACGGATCCGGGGCATTCGAG Pathway CGGAACCGCCCGCCGTGGGAGTTTTTCCAGCOperon GAGCATTCGAGAGTTTTTCAAGGCGGCTTCG AGGGGTTATTCCGTAACGCCGCCGACATGATCTGTCCCAGAATCTCCGCCGCTGTTCGTAGA GCGCCGATGCAGGGTCGGCATCAATCATTCTTGGAGGAGACACatgagccagatccacaagcacaccatcccggccaacatcgccgaccgctgcctgatcaacccgcagcagtacgaggccatgtaccagcagagcatcaacgtcccggacaccttctggggcgagcagggcaagatcctggactggatcaagccgtaccagaaggtcaagaacaccagcttcgccccgggcaacgtcagcatcaagtggtacgaggacggcaccctgaacctggccgccaactgcctggaccgccacctgcaggagaacggcgaccgcaccgccatcatctgggagggcgacgacgccagccagagcaagcacatcagctacaaggagctgcaccgcgacgtctgccgcttcgccaacaccctgctggagctgggcatcaagaagggcgacgtcgtcgccatctacatgccgatggtcccggaggccgccgtcgccatgctggcctgcgcccgcatcggcgccgtccacagcgtcatcttcggcggcttcagcccggaggccgtcgccggccgcatcatcgacagcaacagccgcctggtcatcaccagcgacgagggcgtccgcgccggccgcagcatcccgctgaagaagaacgtcgacgacgccctgaagaacccgaacgtcaccagcgtcgagcacgtcgtcgtcctgaagcgcaccggcggcaagatcgactggcaggagggccgcgacctgtggtggcacgacctggtcgagcaggccagcgaccagcaccaggccgaggagatgaacgccgaggacccgctgttcatcctgtacaccagcggcagcaccggcaagccgaagggcgtcctgcacaccaccggcggctacctggtctacgccgccctgaccttcaagtacgtcttcgactaccacccgggcgacatctactggtgcaccgccgacgtcggctgggtcaccggccacagctacctgctgtacggcccgctggcctgcggcgccaccaccctgatgttcgagggcgtcccgaactggccgaccccggcccgcatggcccaggtcgtcgacaagcaccaggtcaacatcctgtacaccgccccgaccgccatccgcgccctgatggccgagggcgacaaggccatcgagggcaccgaccgcagcagcctgcgcatcctgggcagcgtcggcgagccgatcaacccggaggcctgggagtggtactggaagaagatcggcaacgagaagtgcccggtcgtcgacacctggtggcagaccgagaccggcggcttcatgatcaccccgctgccgggcgccaccgagctgaaggccggcagcgccacccgcccgttcttcggcgtccagccggccctggtcgacaacgagggcaacccgctggagggcgccaccgagggcagcctggtcatcaccgacagctggccgggccaggcccgcaccctgttcggcgaccacgagcgcttcgagcagacctacttcagcaccttcaagaacatgtacttcagcggcgacggcgcccgccgcgacgaggacggctactactggatcaccggccgcgtcgacgacgtcctgaacgtcagcggccaccgcctgggcaccgccgagatcgagagcgccctggtcgcccacccgaagatcgccgaggccgccgtcgtcggcatcccgcacaacatcaagggccaggccatctacgcctacgtcaccctgaaccacggcgaggagccgagcccggagctgtacgccgaggtccgcaactgggtccgcaaggagatcggcccgctggccaccccggacgtcctgcactggaccgacagcctgccgaagacccgcagcggcaagatcatgcgccgcatcctgcgcaagatcgccgccggcgacaccagcaacctgggcgacaccagcaccctggccgacccgggcgtcgtcgagaagctgctggaggagaagcaggccatcgccatgccgagctgaTCATTCTTGGAGGAGACACatgaagcgcgtcctgaccgccctggccgccaccctgccgttcgccgccaacgccgccgacgccatcagcggcgccgtcgagcgccagccgaccaactggcaggccatcatcatgttcctgatcttcgtcgtcttcaccctgggcatcacctactgggccagcaagcgcgtccgcagccgcagcgactactacaccgccggcggcaacatcaccggcttccagaacggcctggccatcgccggcgactacatgagcgccgccagcttcctgggcatcagcgccctggtcttcaccagcggctacgacggcctgatctacagcctgggcttcctggtcggctggccgatcatcctgttcctgatcgccgagcgcctgcgcaacctgggccgctacaccttcgccgacgtcgccagctaccgcctgaagcagggcccgatccgcatcctgagcgcctgcggcagcctggtcgtcgtcgccctgtacctgatcgcccagatggtcggcgccggcaagctgatcgagctgctgttcggcctgaactaccacatcgccgtcgtcctggtcggcgtcctgatgatgatgtacgtcctgttcggcggcatgctggccaccacctgggtccagatcatcaaggccgtcctgctgctgttcggcgccagcttcatggccttcatggtcatgaagcacgtcggcttcagcttcaacaacctgttcagcgaggccatggccgtccacccgaagggcgtcgacatcatgaagccgggcggcctggtcaaggacccgatcagcgccctgagcctgggcctgggcctgatgttcggcaccgccggcctgccgcacatcctgatgcgcttcttcaccgtcagcgacgcccgcgaggcccgcaagagcgtcttctacgccaccggcttcatgggctacttctacatcctgaccttcatcatcggcttcggcgccatcatgctggtcggcgccaacccggagtacaaggacgccgccggccacctgatcggcggcaacaacatggccgccgtccacctggccaacgccgtcggcggcaacctgttcctgggcttcatcagcgccgtcgccttcgccaccatcctggccgtcgtcgccggcctgaccctggccggcgccagcgccgtcagccacgacctgtacgccaacgtcttcaagaagggcgccaccgagcgcgaggagctgcgcgtcagcaagatcaccgtcctgatcctgggcgtcatcgccatcatcctgggcgtcctgttcgagaaccagaacatcgccttcatggtcggcctggccttcgccatcgccgccagctgcaacttcccgatcatcctgctgagcatgtactggagcaagctgaccacccgcggcgccatgatgggcggctggctgggcctgatcaccgccgtcgtcctgatgatcctgggcccgaccatctgggtccagatcctgggccacgagaaggccatcttcccgtacgagtacccggccctgttcagcatcaccgtcgccttcctgggcatctggttcttcagcgccaccgacaacagcgccgagggcgcccgcgagcgcgagctgttccgcgcccagttcatccgcagccagaccggcttcggcgtcgagcagggccgcg cccactga

The synthetic operon is then cloned and transformed as described above.Transformation is confirmed by resistance of the cells to antibioticselection, and gene expression is confirmed by northern blot (to confirmRNA transcription), western blot, or ELISA methods (to confirm proteinexpression).

Growth on Acetate as a Sole Carbon Source. Recombinant M. capsulatustransformed with a vector containing the synthetic operon encoding genesfor acetate utilization are inoculated into 100 mL shake flaskscontaining 20-50 mL NMS media, 1% sodium acetate and 10 ug/mL kanamycin.The flasks are then shaken continuously while being incubated at 42° C.Growth is confirmed by monitoring optical density of the culture overtime. Note that because acetate is the only carbon source provided tothe cells, all cell mass produced must have been derived from acetate.

Example 6 Recombinant Methylomonas Methanica Engineered to Grow onAcetate

Growth and Conjugations. The procedure for growth and conjugation ofMethylomonas methanica was performed essentially identically to theprocedures described for M. capsulatus (above).

Introduction of Acetate Utilization Pathway. Nucleic acid sequencesencoding AcsA (acetyl-CoA synthase) and ActP from E. coli were codonoptimized for expression in M. methanica. The codon optimized nucleicacids encoding AcsA and ActP are synthesized as an operon (SEQ IDNO:124; see Table 19 for components of operon) under control of an hpspromoter with appropriate intergenic regions (CAPITALIZED sequence)incorporating ribosome binding sequences.

TABLE 19Acetate Utilization Pathway Operon Codon Optimized for M. methanica.SEQ ID Gene NO: # Name Nucleotide Sequence SEQ ID HPSTTCGGAATCCCTGACGGGAATTGGCCCGAAGAA NO: 120 promoterGGCAGATGCCATCGTTCAGTATCGAAAGGAACA TGGGGATTTTCAGTCATTGAAGGATCTGGAGAATGTCAGCGGCATTGGCGAGAAAACCCTTCAGGC CAATGAAAAAGACATTCGCTTCACGGATGATTTGAGCGATAAGTCATCCGCGGAAAAAGGTGCGGT AGCTGTGGATAAAAAAGGCGCCAGATAGTAAGCGCTAAGGATTGGGGTGCGTCGCCGGTCGCGGCG GCGCTCCTCGACGGCAGAGTTGGTGCCAGGTTGGCGGATGATTGATGCCGAATATTACGCGACCAA TTCTCGAGGCAAATGAACTGTGAGCTACTGAGTTGCAGGCATTGACAGCCATCCCATTTCTATCATA CAGTTACGGACGCATCACGAGTAGGTGATAAGCCTAGCAGATTGCGGCAGTTGGCAAAATCAGCTA TTACTAATAATTAAAAACTTTCGGAGCACATCACSEQ ID AcsA atgagccaaatccacaaacacaccatcccggcgaacatcgcggatcgctgcttNO: 121 gatcaacccgcaacaatatgaagcgatgtatcaacaaagcatcaacgtcccggataccttctggggcgaacaaggcaaaatcttggattggatcaaaccgtatcaaaaagtcaaaaacaccagcttcgcgccgggcaacgtcagcatcaaatggtatgaagatggcaccttgaacttggcggcgaactgcttggatcgccacttgcaagaaaacggcgatcgcaccgcgatcatctgggaaggcgatgatgcgagccaaagcaaacacatcagctataaagaattgcaccgcgatgtctgccgcttcgcgaacaccttgttggaattgggcatctaaaaaggcgatgtcgtcgcgatctatatgccgatggtcccggaagcggcggtcgcgatgttggcgtgcgcgcgcatcggcgcggtccacagcgtcatcttcggcggcttcagcccggaagcggtcgcgggccgcatcatcgatagcaacagccgcttggtcatcaccagcgatgaaggcgtccgcgcgggccgcagcatcccgttgaaaaaaaacgtcgatgatgcgttgaaaaacccgaacgtcaccagcgtcgaacacgtcgtcgtcttgaaacgcaccggcggcaaaatcgattggcaagaaggccgcgatttgtggtggcacgatttggtcgaacaagcgagcgatcaacaccaagcggaagaaatgaacgcggaagatccgttgttcatcttgtataccagcggcagcaccggcaaaccgaaaggcgtcttgcacaccaccggcggctatttggtctatgcggcgttgaccttcaaatatgtcttcgattatcacccgggcgatatctattggtgcaccgcggatgtcggctgggtcaccggccacagctatttgttgtatggcccgttggcgtgcggcgcgaccaccttgatgttcgaaggcgtcccgaactggccgaccccggcgcgcatggcgcaagtcgtcgataaacaccaagtcaacatcttgtataccgcgccgaccgcgatccgcgcgttgatggcggaaggcgataaagcgatcgaaggcaccgatcgcagcagcttgcgcatcttgggcagcgtcggcgaaccgatcaacccggaagcgtgggaatggtattggaaaaaaatcggcaacgaaaaatgcccggtcgtcgatacctggtggcaaaccgaaaccggcggcttcatgatcaccccgttgccgggcgcgaccgaattgaaagcgggcagcgcgacccgcccgttcttcggcgtccaaccggcgttggtcgataacgaaggcaacccgttggaaggcgcgaccgaaggcagcttggtcatcaccgatagctggccgggccaagcgcgcaccttgttcggcgatcacgaacgcttcgaacaaacctatttcagcaccttcaaaaacatgtatttcagcggcgatggcgcgcgccgcgatgaagatggctattattggatcaccggccgcgtcgatgatgtcttgaacgtcagcggccaccgcttgggcaccgcggaaatcgaaagcgcgttggtcgcgcacccgaaaatcgcggaagcggcggtcgtcggcatcccgcacaacatcaaaggccaagcgatctatgcgtatgtcaccttgaaccacggcgaagaaccgatcccggaattgtatgcggaagtccgcaactgggtccgcaaagaaatcggcccgttggcgaccccggatgtcttgcactggaccgatagcttgccgaaaacccgcagcggcaaaatcatgcgccgcatcttgcgcaaaatcgcggcgggcgataccagcaacttgggcgataccagcaccttggcggatccgggcgtcgtcgaaaaattgttggaagaaaaacaagcgatcgcgatgccgagctaa SEQ ID IntergenicTAATAATTAAAAACTTTCGGAGCACATCAC NO: 122 Region SEQ ID ActPatgaaacgcgtcttgaccgcgttggcggcgaccttgccgttcgcggcgaacgc NO: 123ggcggatgcgatcagcggcgcggtcgaacgccaaccgaccaactggcaagcgatcatcatgttcttgatcttcgtcgtcttcaccttgggcatcacctattgggcgagcaaacgcgtccgcagccgcagcgattattataccgcgggcggcaacatcaccggcttccaaaacggcttggcgatcgcgggcgattatatgagcgcggcgagcttcttgggcatcagcgcgttggtcttcaccagcggctatgatggcttgatctatagcttgggcttcttggtcggctggccgatcatcttgttcttgatcgcggaacgcttgcgcaacttgggccgctataccttcgcggatgtcgcgagctatcgcttgaaacaaggcccgatccgcatcttgagcgcgtgcggcagcttggtcgtcgtcgcgttgtatttgatcgcgcaaatggtcggcgcgggcaaattgatcgaattgttgttcggcttgaactatcacatcgcggtcgtcttggtcggcgtcttgatgatgatgtatgtcttgttcggcggcatgttggcgaccacctgggtccaaatcatcaaagcggtcttgttgttgttcggcgcgagcttcatggcgttcatggtcatgaaacacgtcggcttcagcttcaacaacttgttcagcgaagcgatggcggtccacccgaaaggcgtcgatatcatgaaaccgggcggcttggtcaaagatccgatcagcgcgttgagcttgggcttgggcttgatgttcggcaccgcgggcttgccgcacatcttgatgcgcttcttcaccgtcagcgatgcgcgcgaagcgcgcaaaagcgtcttctatgcgaccggcttcatgggctatttctatatcttgaccttcatcatcggcttcggcgcgatcatgttggtcggcgcgaacccggaatataaagatgcggcgggccacttgatcggcggcaacaacatggcggcggtccacttggcgaacgcggtcggcggcaacttgttcttgggcttcatcagcgcggtcgcgttcgcgaccatcttggcggtcgtcgcgggcttgaccttggcgggcgcgagcgcggtcagccacgatttgtatgcgaacgtcttcaaaaaaggcgcgaccgaacgcgaagaattgcgcgtcagcaaaatcaccgtcttgatcttgggcgtcatcgcgatcatcttgggcgtcttgttcgaaaaccaaaacatcgcgttcatggtcggcttggcgttcgcgatcgcggcgagctgcaacttcccgatcatcttgttgagcatgtattggagcaaattgaccacccgcggcgcgatgatgggcggctggttgggcttgatcaccgcggtcgtcttgatgatcttgggcccgaccatctgggtccaaatcttgggccacgaaaaagcgatcttcccgtatgaatatccggcgttgttcagcatcaccgtcgcgttcttgggcatctggttcttcagcgcgaccgataacagcgcggaaggcgcgcgcgaacgcgaattgttccgcgcgcaattcatccgcagccaaaccggcttcggcgtcgaacaaggccgcgcgcactaa SEQ ID Acetate TTCGGAATCCCTGACGGGAATTGGCCCGAAGAANO: 124 Utilization GGCAGATGCCATCGTTCAGTATCGAAAGGAACA PathwayTGGGGATTTTCAGTCATTGAAGGATCTGGAGAA OperonTGTCAGCGGCATTGGCGAGAAAACCCTTCAGGC CAATGAAAAAGACATTCGCTTCACGGATGATTTGAGCGATAAGTCATCCGCGGAAAAAGGTGCGGT AGCTGTGGATAAAAAAGGCGCCAGATAGTAAGCGCTAAGGATTGGGGTGCGTCGCCGGTCGCGGCG GCGCTCCTCGACGGCAGAGTTGGTGCCAGGTTGGCGGATGATTGATGCCGAATATTACGCGACCAA TTCTCGAGGCAAATGAACTGTGAGCTACTGAGTTGCAGGCATTGACAGCCATCCCATTTCTATCATA CAGTTACGGACGCATCACGAGTAGGTGATAAGCCTAGCAGATTGCGGCAGTTGGCAAAATCAGCTA TTACTAATAATTAAAAACTTTCGGAGCACATCACatgagccaaatccacaaacacaccatcccggcgaacatcgcggatcgctgcttgatcaacccgcaacaatatgaagcgatgtatcaacaaagcatcaacgtcccggataccttctggggcgaacaaggcaaaatcttggattggatcaaaccgtatcaaaaagtcaaaaacaccagcttcgcgccgggcaacgtcagcatcaaatggtatgaagatggcaccttgaacttggcggcgaactgcttggatcgccacttgcaagaaaacggcgatcgcaccgcgatcatctgggaaggcgatgatgcgagccaaagcaaacacatcagctataaagaattgcaccgcgatgtctgccgcttcgcgaacaccttgttggaattgggcatcaaaaaaggcgatgtcgtcgcgatctatatgccgatggtcccggaagcggcggtcgcgatgttggcgtgcgcgcgcatcggcgcggtccacagcgtcatcttcggcggcttcagcccggaagcggtcgcgggccgcatcatcgatagcaacagccgcttggtcatcaccagcgatgaaggcgtccgcgcgggccgcagcatcccgttgaaaaaaaacgtcgatgatgcgttgaaaaacccgaacgtcaccagcgtcgaacacgtcgtcgtcttgaaacgcaccggcggcaaaatcgattggcaagaaggccgcgatttgtggtggcacgatttggtcgaacaagcgagcgatcaacaccaagcggaagaaatgaacgcggaagatccgttgttcatcttgtataccagcggcagcaccggcaaaccgaaaggcgtcttgcacaccaccggcggctatttggtctatgcggcgttgaccttcaaatatgtcttcgattatcacccgggcgatatctattggtgcaccgcggatgtcggctgggtcaccggccacagctatttgttgtatggcccgttggcgtgcggcgcgaccaccttgatgttcgaaggcgtcccgaactggccgaccccggcgcgcatggcgcaagtcgtcgataaacaccaagtcaacatcttgtataccgcgccgaccgcgatccgcgcgttgatggcggaaggcgataaagcgatcgaaggcaccgatcgcagcagcttgcgcatcttgggcagcgtcggcgaaccgatcaacccggaagcgtgggaatggtattggaaaaaaatcggcaacgaaaaatgcccggtcgtcgatacctggtggcaaaccgaaaccggcggcttcatgatcaccccgttgccgggcgcgaccgaattgaaagcgggcagcgcgacccgcccgttcttcggcgtccaaccggcgttggtcgataacgaaggcaacccgttggaaggcgcgaccgaaggcagcttggtcatcaccgatagctggccgggccaagcgcgcaccttgttcggcgatcacgaacgcttcgaacaaacctatttcagcaccttcaaaaacatgtatttcagcggcgatggcgcgcgccgcgatgaagatggctattattggatcaccggccgcgtcgatgatgtcttgaacgtcagcggccaccgcttgggcaccgcggaaatcgaaagcgcgttggtcgcgcacccgaaaatcgcggaagcggcggtcgtcggcatcccgcacaacatcaaaggccaagcgatctatgcgtatgtcaccttgaaccacggcgaagaaccgagcccggaattgtatgcggaagtccgcaactgggtccgcaaagaaatcggcccgttggcgaccccggatgtcttgcactggaccgatagcttgccgaaaacccgcagcggcaaaatcatgcgccgcatcttgcgcaaaatcgcggcgggcgataccagcaacttgggcgataccagcaccttggcggatccgggcgtcgtcgaaaaattgttggaagaaaaacaagcgatcgcgatgccgagctaaTAATAATTAAAAACTTTCGGAGCACATCACatgaaacgcgtcttgaccgcgttggcggcgaccttgccgttcgcggcgaacgcggcggatgcgatcagcggcgcggtcgaacgccaaccgaccaactggcaagcgatcatcatgttcttgatcttcgtcgtcttcaccttgggcatcacctattgggcgagcaaacgcgtccgcagccgcagcgattattataccgcgggcggcaacatcaccggcttccaaaacggcttggcgatcgcgggcgattatatgagcgcggcgagcttcttgggcatcagcgcgttggtcttcaccagcggctatgatggcttgatctatagcttgggcttcttggtcggctggccgatcatcttgttcttgatcgcggaacgcttgcgcaacttgggccgctataccttcgcggatgtcgcgagctatcgcttgaaacaaggcccgatccgcatcttgagcgcgtgcggcagcttggtcgtcgtcgcgttgtatttgatcgcgcaaatggtcggcgcgggcaaattgatcgaattgttgttcggcttgaactatcacatcgcggtcgtcttggtcggcgtcttgatgatgatgtatgtcttgttcggcggcatgttggcgaccacctgggtccaaatcatcaaagcggtcttgttgttgttcggcgcgagcttcatggcgttcatggtcatgatacacgtcggcttcagcttcaacaacttgttcagcgaagcgatggcggtccacccgaaaggcgtcgatatcatgaaaccgggcggcttggtcaaagatccgatcagcgcgttgagcttgggcttgggcttgatgttcggcaccgcgggcttgccgcacatcttgatgcgcttcttcaccgtcagcgatgcgcgcgaagcgcgcaaaagcgtcttctatgcgaccggcttcatgggctatttctatatcttgaccttcatcatcggcttcggcgcgatcatgttggtcggcgcgaacccggaatataaagatgcggcgggccacttgatcggcggcaacaacatggcggcggtccacttggcgaacgcggtcggcggcaacttgttcttgggcttcatcagcgcggtcgcgttcgcgaccatcttggcggtcgtcgcgggcttgaccttggcgggcgcgagcgcggtcagccacgatttgtatgcgaacgtcttcaaaaaaggcgcgaccgaacgcgaagaattgcgcgtcagcaaaatcaccgtcttgatcttgggcgtcatcgcgatcatcttgggcgtcttgttcgaaaaccaaaacatcgcgttcatggtcggcttggcgttcgcgatcgcggcgagctgcaacttcccgatcatcttgttgagcatgtattggagcaaattgaccacccgcggcgcgatgatgggcggctggttgggcttgatcaccgcggtcgtcttgatgatcttgggcccgaccatctgggtccaaatcttgggccacgaaaaagcgatcttcccgtatgaatatccggcgttgttcagcatcaccgtcgcgttcttgggcatctggttcttcagcgcgaccgataacagcgcggaaggcgcgcgcgaacgcgaattgttccgcgcgcaattcatccgcagccaaaccggcttcggcgtcgaacaaggccgc gcgcactaa

The synthetic operon is then cloned and transformed into M. methanica asdescribed above. Transformation is confirmed by resistance of the cellsto antibiotic selection, and gene expression is confirmed by northernblot (to confirm RNA transcription), western blot, or ELISA methods (toconfirm protein expression).

Growth on Acetate as a Sole Carbon Source. Recombinant M. methanicatransformed with a vector containing the synthetic operon encoding genesfor acetate utilization are inoculated into 100 mL shake flaskscontaining 20-50 mL NMS media, 1% sodium acetate and 10 ug/mL kanamycin.The flasks are then shaken continuously while being incubated at 30° C.Growth is confirmed by monitoring optical density of the culture overtime. Note that because acetate is the only carbon source provided tothe cells, all cell mass produced must have been derived from acetate.

Example 7 Recombinant Methylosinus trichosporium Engineered to Grow onLactate

M. trichosporium cells are cultured and conjugated as described above.

Introduction of Lactate Utilization Pathway. Nucleic acid sequencesencoding lactate dehydrogenase D (LdhD) and a lactate permease (LctP)from E. coli were codon optimized for expression in M. trichosporium.The codon optimized nucleic acids encoding LdhD and LctP are synthesizedas an operon (SEQ ID NO:129; see Table 20 for components of operon)under control of an mdh promoter with appropriate intergenic regions(CAPITALIZED sequence) incorporating ribosome binding sequences.

TABLE 20Lactate Utilization Pathway Operon Codon Optimized for M. trichosporiumSEQ ID NO: # Gene Name Nucleotide Sequence SEQ ID MDHTTTGCCTCGATCGGCGGTCCTTGTGACAGGGAG NO: 125 promoterATATTCCCGACGGATCCGGGGCATTCGAGCGG AACCGCCCGCCGTGGGAGTTTTTCCAGCGAGCATTCGAGAGTTTTTCAAGGCGGCTTCGAGGGGTT ATTCCGTAACGCCGCCGACATGATCTGTCCCAGAATCTCCGCCGCTGTTCGTAGAGCGCCGATGCA GGGTCGGCATCAATCATTCTTGGAGGAGACACSEQ ID LdhD atgaagctggcggtctattcgaccaagcagtatgacaagaagtatctgcagcaNO: 126 ggtcaacgagtcgttcggcttcgagctggagttcttcgacttcctgctgaccgagaagaccgcgaagaccgcgaacggctgcgaggcggtctgcatcttcgtcaacgacgacggctcgcgcccggtcctggaggagctgaagaagcatggcgtcaagtatatcgcgctgcgctgcgcgggcttcaacaacgtcgacctggacgcggcgaaggagctgggcctgaaggtcgtccgcgtcccggcgtatgacccggaggcggtcgcggagcatgcgatcggcatgatgatgaccctgaaccgccgcatccatcgcgcgtatcagcgcacccgcgacgcgaacttctcgctggagggcctgaccggcttcaccatgtatggcaagaccgcgggcgtcatcggcaccggcaagatcggcgtcgcgatgctgcgcatcctgaagggcttcggcatgcgcctgctggcgttcgacccgtatccgtcggcggcggcgctggagctgggcgtcgagtatgtcgacctgccgaccctgttctcggagtcggacgtcatctcgctgcattgcccgctgaccccggagaactatcatctgctgaacgaggcggcgttcgagcagatgaagaacggcgtcatgatcgtcaacacctcgcgcggcgcgctgatcgactcgcaggcggcgatcgaggcgctgaagaaccagaagatcggctcgctgggcatggacgtctatgagaacgagcgcgacctgttcttcgaggacaagtcgaacgacgtcatccaggacgacgtcttccgccgcctgtcggcgtgccataacgtcctgttcaccggccatcaggcgttcctgaccgcggaggcgctgacctcgatctcgcagaccaccctgcagaacctgtcgaacctggagaagggcgagacctgcccgaacg agctggtctga SEQ IDIntergenic TCATTCTTGGAGGAGACAC NO: 127 Region SEQ ID LctPatgaacctgtggcagcagaactatgacccggcgggcaacatctggctgtcgt NO: 128cgctgatcgcgtcgctgccgatcctgttcttcttcttcgcgctgatcaagctgaagctgaagggctatgtcgcggcgtcgtggaccgtcgcgatcgcgctggcggtcgcgctgctgttctataagatgccggtcgcgaacgcgctggcgtcggtcgtctatggcttcttctatggcctgtggccgatcgcgtggatcatcatcgcggcggtcttcgtctataagatctcggtcaagaccggccagttcgacatcatccgctcgtcgatcctgtcgatcaccccggaccagcgcctgcagatgctgatcgtcggcttctgcttcggcgcgttcctggagggcgcggcgggcttcggcgcgccggtcgcgatcaccgcggcgctgctggtcggcctgggcttcaagccgctgtatgcggcgggcctgtgcctgatcgtcaacaccgcgccggtcgcgttcggcgcgatgggcatcccgatcctggtcgcgggccaggtcaccggcatcgactcgttcgagatcggccagatggtcggccgccagctgccgttcatgaccatcatcgtcctgttctggatcatggcgatcatggacggctggcgcggcatcaaggagacctggccggcggtcgtcgtcgcgggcggctcgttcgcgatcgcgcagtatctgtcgtcgaacttcatcggcccggagctgccggacatcatctcgtcgctggtctcgctgctgtgcctgaccctgttcctgaagcgctggcagccggtccgcgtcttccgcttcggcgacctgggcgcgtcgcaggtcgacatgaccctggcgcataccggctataccgcgggccaggtcctgcgcgcgtggaccccgttcctgttcctgaccgcgaccgtcaccctgtggtcgatcccgccgttcaaggcgctgttcgcgtcgggcggcgcgctgtatgagtgggtcatcaacatcccggtcccgtatctggacaagctggtcgcgcgcatgccgccggtcgtctcggaggcgaccgcgtatgcggcggtcttcaagttcgactggttctcggcgaccggcaccgcgatcctgttcgcggcgctgctgtcgatcgtctggctgaagatgaagccgtcggacgcgatctcgaccttcggctcgaccctgaaggagctggcgctgccgatctattcgatcggcatggtcctggcgttcgcgttcatctcgaactattcgggcctgtcgtcgaccctggcgctggcgctggcgcataccggccatgcgttcaccttcttctcgccgttcctgggctggctgggcgtcttcctgaccggctcggacacctcgtcgaacgcgctgttcgcggcgctgcaggcgaccgcggcgcagcagatcggcgtctcggacctgctgctggtcgcggcgaacaccaccggcggcgtcaccggcaagatgatctcgccgcagtcgatcgcgatcgcgtgcgcggcggtcggcctggtcggcaaggagtcggacctgttccgcttcaccgtcaagcattcgctgatcttcacctgcatcgtcggcgtcatcaccaCCctgcaggcgtatgtcctgacctggatgatcccgtga SEQ ID LactateTTTGCCTCGATCGGCGGTCCTTGTGACAGGGAG NO: 129 UtilizationATATTCCCGACGGATCCGGGGCATTCGAGCGG PathwayAACCGCCCGCCGTGGGAGTTTTTCCAGCGAGCA OperonTTCGAGAGTTTTTCAAGGCGGCTTCGAGGGGTT ATTCCGTAACGCCGCCGACATGATCTGTCCCAGAATCTCCGCCGCTGTTCGTAGAGCGCCGATGCA GGGTCGGCATCAATCATTCTTGGAGGAGACACatgaagctggcggtctattcgaccaagcagtatgacaagaagtatctgcagcaggtcaacgagtcgttcggcttcgagctggagttcttcgacttcctgctgaccgagaagaccgcgaagaccgcgaacggctgcgaggcggtctgcatcttcgtcaacgacgacggctcgcgcccggtcctggaggagctgaagaagcatggcgtcaagtatatcgcgctgcgctgcgcgggcttcaacaacgtcgacctggacgcggcgaaggagctgggcctgaaggtcgtccgcgtcccggcgtatgacccggaggcggtcgcggagcatgcgatcggcatgatgatgaccctgaaccgccgcatccatcgcgcgtatcagcgcacccgcgacgcgaacttctcgctggagggcctgaccggcttcaccatgtatggcaagaccgcgggcgtcatcggcaccggcaagatcggcgtcgcgatgctgcgcatcctgaagggcttcggcatgcgcctgctggcgttcgacccgtatccgtcggcggcggcgctggagctgggcgtcgagtatgtcgacctgccgaccctgttctcggagtcggacgtcatctcgctgcattgcccgctgaccccggagaactatcatctgctgaacgaggcggcgttcgagcagatgaagaacggcgtcatgatcgtcaacacctcgcgcggcgcgctgatcgactcgcaggcggcgatcgaggcgctgaagaaccagaagatcggctcgctgggcatggacgtctatgagaacgagcgcgacctgttcttcgaggacaagtcgaacgacgtcatccaggacgacgtcttccgccgcctgtcggcgtgccataacgtcctgttcaccggccatcaggcgttcctgaccgcggaggcgctgacctcgatctcgcagaccaccctgcagaacctgtcgaacctggagaagggcgagacctgcccgaacgagctggtctgaTCATTCTTGGAGGAGACACatgaacctgtggcagcagaactatgacccggcgggcaacatctggctgtcgtcgctgatcgcgtcgctgccgatcctgttcttcttcttcgcgctgatcaagctgaagctgaagggctatgtcgcggcgtcgtggaccgtcgcgatcgcgctggcggtcgcgctgctgttctataagatgccggtcgcgaacgcgctggcgtcggtcgtctatggcttcttctatggcctgtggccgatcgcgtggatcatcatcgcggcggtcttcgtctataagatctcggtcaagaccggccagttcgacatcatccgctcgtcgatcctgtcgatcaccccggaccagcgcctgcagatgctgatcgtcggcttctgcttcggcgcgttcctggagggcgcggcgggcttcggcgcgccggtcgcgatcaccgcggcgctgctggtcggcctgggcttcaagccgctgtatgcggcgggcctgtgcctgatcgtcaacaccgcgccggtcgcgttcggcgcgatgggcatcccgatcctggtcgcgggccaggtcaccggcatcgactcgttcgagatcggccagatggtcggccgccagctgccgttcatgaccatcatcgtcctgttctggatcatggcgatcatggacggctggcgcggcatcaaggagacctggccggcggtcgtcgtcgcgggcggctcgttcgcgatcgcgcagtatctgtcgtcgaacttcatcggcccggagctgccggacatcatctcgtcgctggtctcgctgctgtgcctgaccctgttcctgaagcgctggcagccggtccgcgtcttccgcttcggcgacctgggcgcgtcgcaggtcgacatgaccctggcgcataccggctataccgcgggccaggtcctgcgcgcgtggaccccgttcctgttcctgaccgcgaccgtcaccctgtggtcgatcccgccgttcaaggcgctgttcgcgtcgggcggcgcgctgtatgagtgggtcatcaacatcccggtcccgtatctggacaagctggtcgcgcgcatgccgccggtcgtctcggaggcgaccgcgtatgcggcggtcttcaagttcgactggttctcggcgaccggcaccgcgatcctgttcgcggcgctgctgtcgatcgtctggctgaagatgaagccgtcggacgcgatctcgaccttcggctcgaccctgaaggagctggcgctgccgatctattcgatcggcatggtcctggcgttcgcgttcatctcgaactattcgggcctgtcgtcgaccctggcgctggcgctggcgcataccggccatgcgttcaccttcttctcgccgttcctgggctggctgggcgtcttcctgaccggctcggacacctcgtcgaacgcgctgttcgcggcgctgcaggcgaccgcggcgcagcagatcggcgtctcggacctgctgctggtcgcggcgaacaccaccggcggcgtcaccggcaagatgatctcgccgcagtcgatcgcgatcgcgtgcgcggcggtcggcctggtcggcaaggagtcggacctgttccgcttcaccgtcaagcattcgctgatcttcacctgcatcgtcggcgtcatcaccaccctgcaggcgtatgtcctga cctggatgatcccgtga

The synthetic operon is then cloned and transformed as described above.Transformation is confirmed by resistance of the cells to antibioticselection, and gene expression is confirmed by northern blot (to confirmRNA transcription), western blot, or ELISA methods (to confirm proteinexpression).

Growth on Lactate as a Sole Carbon Source. Recombinant M. trichosporiumtransformed with a vector containing the synthetic operon encoding genesfor lactate utilization are inoculated into 100 mL shake flaskscontaining 20-50 mL NMS media, 1% sodium lactate and 10 ug/mL kanamycin.The flasks are then shaken continuously while being incubated at 30° C.Growth is confirmed by monitoring optical density of the culture overtime. Note that because lactate is the only carbon source provided tothe cells, all cell mass produced must have been derived from lactate.

Example 8 Recombinant Methylococcus Capsulatus Bath Engineered to Growon Lactate

M. capsulatus cells are cultured and conjugated as described above.

Introduction of Lactate Utilization Pathway. Nucleic acid sequencesencoding lactate dehydrogenase D (LdhD) and a lactate permease (LctP)from E. coli were codon optimized for expression in M. trichosporium.The codon optimized nucleic acids encoding LdhD and LctP are synthesizedas an operon (SEQ ID NO:134; see Table 21 for components of operon)under control of an mdh promoter with appropriate intergenic regions(CAPITALIZED sequence) incorporating ribosome binding sequences.

TABLE 21Lactate Utilization Pathway Operon Codon Optimized for M. capsulatusSEQ ID Gene NO: # Name Nucleotide Sequence SEQ ID MDHTTTGCCTCGATCGGCGGTCCTTGTGACAGGGAG NO: 130 promoterATATTCCCGACGGATCCGGGGCATTCGAGCGGA ACCGCCCGCCGTGGGAGTTTTTCCAGCGAGCATTCGAGAGTTTTTCAAGGCGGCTTCGAGGGGTTA TTCCGTAACGCCGCCGACATGATCTGTCCCAGAATCTCCGCCGCTGTTCGTAGAGCGCCGATGCAG GGTCGGCATCAATCATTCTTGGAGGAGACAC SEQ IDLdhD atgaagctggccgtctacagcaccaagcagtacgacaagaagtacctgcagc NO: 131aggtcaacgagagcttcggcttcgagctggagttcttcgacttcctgctgaccgagaagaccgccaagaccgccaacggctgcgaggccgtctgcatcttcgtcaacgacgacggcagccgcccggtcctggaggagctgaagaagcacggcgtcaagtacatcgccctgcgctgcgccggcttcaacaacgtcgacctggacgccgccaaggagctgggcctgaaggtcgtccgcgtcccggcctacgacccggaggccgtcgccgagcacgccatcggcatgatgatgaccctgaaccgccgcatccaccgcgcctaccagcgcacccgcgacgccaacttcagcctggagggcctgaccggcttcaccatgtacggcaagaccgccggcgtcatcggcaccggcaagatcggcgtcgccatgctgcgcatcctgaagggcttcggcatgcgcctgctggccttcgacccgtacccgagcgccgccgccctggagctgggcgtcgagtacgtcgacctgccgaccctgttcagcgagagcgacgtcatcagcctgcactgcccgctgaccccggagaactaccacctgctgaacgaggccgccttcgagcagatgaagaacggcgtcatgatcgtcaacaccagccgcggcgccctgatcgacagccaggccgccatcgaggccctgaagaaccagaagatcggcagcctgggcatggacgtctacgagaacgagcgcgacctgttcttcgaggacaagagcaacgacgtcatccaggacgacgtcttccgccgcctgagcgcctgccacaacgtcctgttcaccggccaccaggccttcctgaccgccgaggccctgaccagcatcagccagaccaccctgcagaacctgagcaacctggagaagggcgagacctgcccgaacg SEQ ID IntergenicTCATTCTTGGAGGAGACAC NO: 132 Region SEQ ID LctPatgaacctgtggcagcagaactacgacccggccggcaacatctggctgagc NO: 133agcctgatcgccagcctgccgatcctgttcttcttcttcgccctgatcaagctgaagctgaagggctacgtcgccgccagctggaccgtcgccatcgccctggccgtcgccctgctgttctacaagatgccggtcgccaacgccctggccagcgtcgtctacggcttcttctacggcctgtggccgatcgcctggatcatcatcgccgccgtcttcgtctacaagatcagcgtcaagaccggccagttcgacatcatccgcagcagcatcctgagcatcaccccggaccagcgcctgcagatgctgatcgtcggcttctgcttcggcgccttcctggagggcgccgccggcttcggcgccccggtcgccatcaccgccgccctgctggtcggcctgggcttcaagccgctgtacgccgccggcctgtgcctgatcgtcaacaccgccccggtcgccttcggcgccatgggcatcccgatcctggtcgccggccaggtcaccggcatcgacagcttcgagatcggccagatggtcggccgccagctgccgttcatgaccatcatcgtcctgttctggatcatggccatcatggacggctggcgcggcatcaaggagacctggccggccgtcgtcgtcgccggcggcagcttcgccatcgcccagtacctgagcagcaacttcatcggcccggagctgccggacatcatcagcagcctggtcagcctgctgtgcctgaccctgttcctgaagcgctggcagccggtccgcgtcttccgcttcggcgacctgggcgccagccaggtcgacatgaccctggcccacaccggctacaccgccggccaggtcctgcgcgcctggaccccgttcctgttcctgaccgccaccgtcaccctgtggagcatcccgccgttcaaggccctgttcgccagcggcggcgccctgtacgagtgggtcatcaacatcccggtcccgtacctggacaagctggtcgcccgcatgccgccggtcgtcagcgaggccaccgcctacgccgccgtcttcaagttcgactggttcagcgccaccggcaccgccatcctgttcgccgccctgctgagcatcgtctggctgaagatgaagccgagcgacgccatcagcaccttcggcagcaccctgaaggagctggccctgccgatctacagcatcggcatggtcctggccttcgccttcatcagcaactacagcggcctgagcagcaccctggccctggccctggcccacaccggccacgccttcaccttcttcagcccgttcctgggctggctgggcgtcttcctgaccggcagcgacaccagcagcaacgccctgttcgccgccctgcaggccaccgccgcccagcagatcggcgtcagcgacctgctgctggtcgccgccaacaccaccggcggcgtcaccggcaagatgatcagcccgcagagcatcgccatcgcctgcgccgccgtcggcctggtcggcaaggagagcgacctgttccgcttcaccgtcaagcacagcctgatcttcacctgcatcgtcggcgtcatcaccaccctgcaggcctacgtcctgacctggatgatcccgtga SEQ ID LactateTTTGCCTCGATCGGCGGTCCTTGTGACAGGGAG NO: 134 UtilizationATATTCCCGACGGATCCGGGGCATTCGAGCGGA PathwayACCGCCCGCCGTGGGAGTTTTTCCAGCGAGCAT OperonTCGAGAGTTTTTCAAGGCGGCTTCGAGGGGTTA TTCCGTAACGCCGCCGACATGATCTGTCCAGAATCTCCGCCGCTGTTCGTAGAGCGCCGATGCAG GGTCGGCATCAATCATTCTTGGAGGAGACACatgaagctggccgtctacagcaccaagcagtacgacaagaagtacctgcagcaggtcaacgagagcttcggcttcgagctggagttcttcgacttcctgctgaccgagaagaccgccaagaccgccaacggctgcgaggccgtctgcatcttcgtcaacgacgacggcagccgcccggtcctggaggagctgaagaagcacggcgtcaagtacatcgccctgcgctgcgccggcttcaacaacgtcgacctggacgccgccaaggagctgggcctgaaggtcgtccgcgtcccggcctacgacccggaggccgtcgccgagcacgccatcggcatgatgatgaccctgaaccgccgcatccaccgcgcctaccagcgcacccgcgacgccaacttcagcctggagggcctgaccggcttcaccatgtacggcaagaccgccggcgtcatcggcaccggcaagatcggcgtcgccatgctgcgcatcctgaagggcttcggcatgcgcctgctggccttcgacccgtacccgagcgccgccgccctggagctgggcgtcgagtacgtcgacctgccgaccctgttcagcgagagcgacgtcatcagcctgcactgcccgctgaccccggagaactaccacctgctgaacgaggccgccttcgagcagatgaagaacggcgtcatgatcgtcaacaccagccgcggcgccctgatcgacagccaggccgccatcgaggccctgaagaaccagaagatcggcagcctgggcatggacgtctacgagaacgagcgcgacctgttcttcgaggacaagagcaacgacgtcatccaggacgacgtcttccgccgcctgagcgcctgccacaacgtcctgttcaccggccaccaggccttcctgaccgccgaggccctgaccagcatcagccagaccaccctgcagaacctgagcaacctggagaagggcgagacctgcccgaacgagctggtctgaTCATTCTTGGAGGAGACACatgaacctgtggcagcagaactacgacccggccggcaacatctggctgagcagcctgatcgccagcctgccgatcctgttcttcttcttcgccctgatcaagctgaagctgaagggctacgtcgccgccagctggaccgtcgccatcgccctggccgtcgccctgctgttctacaagatgccggtcgccaacgccctggccagcgtcgtctacggcttcttctacggcctgtggccgatcgcctggatcatcatcgccgccgtcttcgtctacaagatcagcgtcaagaccggccagttcgacatcatccgcagcagcatcctgagcatcaccccggaccagcgcctgcagatgctgatcgtcggcttctgcttcggcgccttcctggagggcgccgccggcttcggcgccccggtcgccatcaccgccgccctgctggtcggcctgggcttcaagccgctgtacgccgccggcctgtgcctgatcgtcaacaccgccccggtcgccttcggcgccatgggcatcccgatcctggtcgccggccaggtcaccggcatcgacagcttcgagatcggccagatggtcggccgccagctgccgttcatgaccatcatcgtcctgttctggatcatggccatcatggacggctggcgcggcatcaaggagacctggccggccgtcgtcgtcgccggcggcagcttcgccatcgcccagtacctgagcagcaacttcatcggcccggagctgccggacatcatcagcagcctggtcagcctgctgtgcctgaccctgttcctgaagcgctggcagccggtccgcgtcttccgcttcggcgacctgggcgccagccaggtcgacatgaccctggcccacaccggctacaccgccggccaggtcctgcgcgcctggaccccgttcctgttcctgaccgccaccgtcaccctgtggagcatcccgccgttcaaggccctgttcgccagcggcggcgccctgtacgagtgggtcatcaacatcccggtcccgtacctggacaagctggtcgcccgcatgccgccggtcgtcagcgaggccaccgcctacgccgccgtcttcaagttcgactggttcagcgccaccggcaccgccatcctgttcgccgccctgctgagcatcgtctggctgaagatgaagccgagcgacgccatcagcaccttcggcagcaccctgaaggagctggccctgccgatctacagcatcggcatggtcctggccttcgccttcatcagcaactacagcggcctgagcagcaccctggccctggccctggcccacaccggccacgccttcaccHcttcagcccgttcctgggctggctgggcgtcttcctgaccggcagcgacaccagcagcaacgccctgttcgccgccctgcaggccaccgccgcccagcagatcggcgtcagcgacctgctgctggtcgccgccaacaccaccggcggcgtcaccggcaagatgatcagcccgcagagcatcgccatcgcctgcgccgccgtcggcctggtcggcaaggagagcgacctgttccgcttcaccgtcaagcacagcctgatcttcacctgcatcgtcggcgtcatcaccaccctgcaggcctacgtcctgacctggatgatcccgtga

The synthetic operon is then cloned and transformed as described above.Transformation is confirmed by resistance of the cells to antibioticselection, and gene expression is confirmed by northern blot (to confirmRNA transcription), western blot, or ELISA methods (to confirm proteinexpression).

Growth on Lactate as a Sole Carbon Source. Recombinant M. capsulatustransformed with a vector containing the synthetic operon encoding genesfor lactate utilization are inoculated into 100 mL shake flaskscontaining 20-50 mL NMS media, 1% sodium lactate and 10 ug/mL kanamycin.The flasks are then shaken continuously while being incubated at 42° C.Growth is confirmed by monitoring optical density of the culture overtime. Note that because lactate is the only carbon source provided tothe cells, all cell mass produced must have been derived from lactate.

Example 9 Methylomonas Methanica Engineered to Grow on Lactate

Growth and Conjugations. The procedure for growth and conjugation ofMethylomonas methanica was performed essentially identically to theprocedures described above for M. capsulatus.

Introduction of Lactate Utilization Pathway. Nucleic acid sequencesencoding lactate dehydrogenase D (LdhD) and a lactate permease (LctP)from E. coli were codon optimized for expression in M. methanica. Thecodon optimized nucleic acids encoding LdhD and LctP are synthesized asan operon (SEQ ID NO:139; see Table 22 for components of operon) undercontrol of an hps promoter with appropriate intergenic regions(CAPITALIZED sequence) incorporating ribosome binding sequences.

TABLE 22Lactate Utilization Pathway Operon Codon Optimized for M. methanicaSEQ ID NO: # Gene Name Nucleotide Sequence SEQ ID HPSTTCGGAATCCCTGACGGGAATTGGCCCGAAG NO: 135 promoterAAGGCAGATGCCATCGTTCAGTATCGAAAGG AACATGGGGATTTTCAGTCATTGAAGGATCTGGAGAATGTCAGCGGCATTGGCGAGAAAACCC TTCAGGCCAATGAAAAAGACATTCGCTTCACGGATGATTTGAGCGATAAGTCATCCGCGGAAA AAGGTGCGGTAGCTGTGGATAAAAAAGGCGCCAGATAGTAAGCGCTAAGGATTGGGGTGCGT CGCCGGTCGCGGCGGCGCTCCTCGACGGCAGAGTTGGTGCCAGGTTGGCGGATGATTGATGCC GAATATTACGCGACCAATTCTCGAGGCAAATGAACTGTGAGCTACTGAGTTGCAGGCATTGACA GCCATCCCATTTCTATCATACAGTTACGGACGCATCACGAGTAGGTGATAAGCCTAGCAGATT GCGGCAGTTGGCAAAATCAGCTATTACTAATAATTAAAAACTTTCGGAGCACATCAC SEQ ID LdhDatgaaattggcggtctatagcaccaaacaatatgataaaaaatatttgcaaca NO: 136agtcaacgaaagcttcggcttcgaattggaattcttcgatttcttgttgaccgaaaaaaccgcgaaaaccgcgaacggctgcgaagcggtctgcatcttcgtcaacgatgatggcagccgcccggtcttggaagaattgaaaaaacacggcgtcaaatatatcgcgttgcgctgcgcgggcttcaacaacgtcgatttggatgcggcgaaagaattgggcttgaaagtcgtccgcgtcccggcgtatgatccggaagcggtcgcggaacacgcgatcggcatgatgatgaccttgaaccgccgcatccaccgcgcgtatcaacgcacccgcgatgcgaacttcagcttggaaggcttgaccggcttcaccatgtatggcaaaaccgcgggcgtcatcggcaccggcaaaatcggcgtcgcgatgttgcgcatcttgaaaggcttcggcatgcgcttgttggcgttcgatccgtatccgagcgcggcggcgttggaattgggcgtcgaatatgtcgatttgccgaccttgttcagcgaaagcgatgtcatcagcttgcactgcccgttgaccccggaaaactatcacttgttgaacgaagcggcgttcgaacaaatgaaaaacggcgtcatgatcgtcaacaccagccgcggcgcgttgatcgatagccaagcggcgatcgaagcgttgaaaaaccaaaaaatcggcagcttgggcatggatgtctatgaaaacgaacgcgatttgttcttcgaagataaaagcaacgatgtcatccaagatgatgtcttccgccgcttgagcgcgtgccacaacgtcttgttcaccggccaccaagcgttcttgaccgcggaagcgttgaccagcatcagccaaaccaccttgcaaaacttgagcaacttggaaaaaggcgaaacctgcc cgaacgaattggtctaaSEQ ID Intergenic TAATAATTAAAAACTTTCGGAGCACATCAC NO: 137 Region SEQ IDLctP atgaacttgtggcaacaaaactatgatccggcgggcaacatctggttgagc NO: 138agcttgatcgcgagcttgccgatcttgttcttcttcttcgcgttgatcaaattgaaattgaaaggctatgtcgcggcgagctggaccgtcgcgatcgcgttggcggtcgcgttgttgttctataaaatgccggtcgcgaacgcgttggcgagcgtcgtctatggcttcttctatggcttgtggccgatcgcgtggatcatcatcgcggcggtcttcgtctataaaatcagcgtcaaaaccggccaattcgatatcatccgcagcagcatcttgagcatcaccccggatcaacgcttgcaaatgttgatcgtcggcttctgcttcggcgcgttcttggaaggcgcggcgggcttcggcgcgccggtcgcgatcaccgcggcgttgttggtcggcttgggcttcaaaccgttgtatgcggcgggcttgtgcttgatcgtcaacaccgcgccggtcgcgttcggcgcgatgggcatcccgatcttggtcgcgggccaagtcaccggcatcgatagcttcgaaatcggccaaatggtcggccgccaattgccgttcatgaccatcatcgtcttgttctggatcatggcgatcatggatggctggcgcggcatcaaagaaacctggccggcggtcgtcgtcgcgggcggcagcttcgcgatcgcgcaatatttgagcagcaacttcatcggcccggaattgccggatatcatcagcagcttggtcagcttgttgtgcttgaccttgttcttgaaacgctggcaaccggtccgcgtcttccgcttcggcgatttgggcgcgagccaagtcgatatgaccttggcgcacaccggctataccgcgggccaagtcttgcgcgcgtggaccccgttcttgttcttgaccgcgaccgtcaccttgtggagcatcccgccgttcaaagcgttgttcgcgagcggcggcgcgttgtatgaatgggtcatcaacatcccggtcccgtatttggataaattggtcgcgcgcatgccgccggtcgtcagcgaagcgaccgcgtatgcggcggtcttcaaattcgattggttcagcgcgaccggcaccgcgatcttgttcgcggcgttgttgagcatcgtctggttgaaaatgaaaccgagcgatgcgatcagcaccttcggcagcaccttgaaagaattggcgttgccgatctatagcatcggcatggtcttggcgttcgcgttcatcagcaactatagcggcttgagcagcaccttggcgttggcgttggcgcacaccggccacgcgttcaccttcttcagcccgttcttgggctggttgggcgtcttcttgaccggcagcgataccagcagcaacgcgttgttcgcggcgttgcaagcgaccgcggcgcaacaaatcggcgtcagcgatttgttgttggtcgcggcgaacaccaccggcggcgtcaccggcaaaatgatcagcccgcaaagcatcgcgatcgcgtgcgcggcggtcggcttggtcggcaaagaaagcgatttgttccgcttcaccgtcaaacacagcttgatcttcacctgcatcgtcggcgtcatcaccaccttgcaagcgtatgtcttgacctggat gatcccgtaa SEQ IDLactate TTCGGAATCCCTGACGGGAATTGGCCCGAAG NO: 139 UtilizationAAGGCAGATGCCATCGTTCAGTATCGAAAGG Pathway AACATGGGGATTTTCAGTCATTGAAGGATCTGOperon GAGAATGTCAGCGGCATTGGCGAGAAAACCC TTCAGGCCAATGAAAAAGACATTCGCTTCACGGATGATTTGAGCGATAAGTCATCCGCGGAAA AAGGTGCGGTAGCTGTGGATAAAAAAGGCGCCAGATAGTAAGCGCTAAGGATTGGGGTGCGT CGCCGGTCGCGGCGGCGCTCCTCGACGGCAGAGTTGGTGCCAGGTTGGCGGATGATTGATGCC GAATATTACGCGACCAATTCTCGAGGCAAATGAACTGTGAGCTACTGAGTTGCAGGCATTGACA GCCATCCCATTTCTATCATACAGTTACGGACGCATCACGAGTAGGTGATAAGCCTAGCAGATT GCGGCAGTTGGCAAAATCAGCTATTACTAATAATTAAAAACTTTCGGAGCACATCACatgaaattggcggtctatagcaccaaacaatatgataaaaaatatttgcaacaagtcaacgaaagcttcggcttcgaattggaattcttcgatttcttgttgaccgaaaaaaccgcgaaaaccgcgaacggctgcgaagcggtctgcatcttcgtcaacgatgatggcagccgcccggtcttggaagaattgaaaaaacacggcgtcaaatatatcgcgttgcgctgcgcgggcttcaacaacgtcgatttggatgcggcgaaagaattgggcttgaaagtcgtccgcgtcccggcgtatgatccggaagcggtcgcggaacacgcgatcggcatgatgatgaccttgaaccgccgcatccaccgcgcgtatcaacgcacccgcgatgcgaacttcagcttggaaggcttgaccggcttcaccatgtatggcaaaaccgcgggcgtcatcggcaccggcaaaatcggcgtcgcgatgttgcgcatcttgaaaggcttcggcatgcgcttgttggcgttcgatccgtatccgagcgcggcggcgttggaattgggcgtcgaatatgtcgatttgccgaccttgttcagcgaaagcgatgtcatcagcttgcactgcccgttgaccccggaaaactatcacttgttgaacgaagcggcgttcgaacaaatgaaaaacggcgtcatgatcgtcaacaccagccgcggcgcgttgatcgatagccaagcggcgatcgaagcgttgaaaaaccaaaaaatcggcagcttgggcatggatgtctatgaaaacgaacgcgatttgttcttcgaagataaaagcaacgatgtcatccaagatgatgtcttccgccgcttgagcgcgtgccacaacgtcttgttcaccggccaccaagcgttcttgaccgcggaagcgttgaccagcatcagccaaaccaccttgcaaaacttgagcaacttggaaaaaggcgaaacctgcccgaacgaattggtctaaTAATAATTAAAAACTTTCGGAGCACATCACatgaacttgtggcaacaaaactatgatccggcgggcaacatctggttgagcagcttgatcgcgagcttgccgatcttgttcttcttcttcgcgttgatcaaattgaaattgaaaggctatgtcgcggcgagctggaccgtcgcgatcgcgttggcggtcgcgttgttgttctataaaatgccggtcgcgaacgcgttggcgagcgtcgtctatggcttcttctatggcttgtggccgatcgcgtggatcatcatcgcggcggtcttcgtctataaaatcagcgtcaaaaccggccaattcgatatcatccgcagcagcatcttgagcatcaccccggatcaacgcttgcaaatgttgatcgtcggcttctgcttcggcgcgttcttggaaggcgcggcgggcttcggcgcgccggtcgcgatcaccgcggcgttgttggtcggcttgggcttcaaaccgttgtatgcggcgggcttgtgcttgatcgtcaacaccgcgccggtcgcgttcggcgcgatgggcatcccgatcttggtcgcgggccaagtcaccggcatcgatagcttcgaaatcggccaaatggtcggccgccaattgccgttcatgaccatcatcgtcttgttctggatcatggcgatcatggatggctggcgcggcatcaaagaaacctggccggcggtcgtcgtcgcgggcggcagcttcgcgatcgcgcaatatttgagcagcaacttcatcggcccggaattgccggatatcatcagcagcttggtcagcttgttgtgcttgaccttgttcttgaaacgctggcaaccggtccgcgtcttccgcttcggcgatttgggcgcgagccaagtcgatatgaccttggcgcacaccggctataccgcgggccaagtcttgcgcgcgtggaccccgttcttgttcttgaccgcgaccgtcaccttgtggagcatcccgccgttcaaagcgttgttcgcgagcggcggcgcgttgtatgaatgggtcatcaacatcccggtcccgtatttggataaattggtcgcgcgcatgccgccggtcgtcagcgaagcgaccgcgtatgcggcggtcttcaaattcgattggttcagcgcgaccggcaccgcgatcttgttcgcggcgttgttgagcatcgtctggttgaaaatgaaaccgagcgatgcgatcagcaccttcggcagcaccttgaaagaattggcgttgccgatctatagcatcggcatggtcttggcgttcgcgttcatcagcaactatagcggcttgagcagcaccttggcgttggcgttggcgcacaccggccacgcgttcaccttcttcagcccgttcttgggctggttgggcgtcttcttgaccggcagcgataccagcagcaacgcgttgttcgcggcgttgcaagcgaccgcggcgcaacaaatcggcgtcagcgatttgttgttggtcgcggcgaacaccaccggcggcgtcaccggcaaaatgatcagcccgcaaagcatcgcgatcgcgtgcgcggcggtcggcttggtcggcaaagaaagcgatttgttccgcttcaccgtcaaacacagcttgatcttcacctgcatcgtcggcgtcatcaccaccttgcaagcgtatgtcttgacctg gatgatcccgtaa

The synthetic operon is then cloned and transformed into M. methanica asdescribed above. Transformation is confirmed by resistance of the cellsto antibiotic selection, and gene expression is confirmed by northernblot (to confirm RNA transcription), western blot, or ELISA methods (toconfirm protein expression).

Growth on Lactate as a Sole Carbon Source. Recombinant M. methanicatransformed with a vector containing the synthetic operon encoding genesfor lactate utilization are inoculated into 100 mL shake flaskscontaining 20-50 mL NMS media, 1% sodium lactate and 10 ug/mL kanamycin.The flasks are then shaken continuously while being incubated at 30° C.Growth is confirmed by monitoring optical density of the culture overtime. Note that because lactate is the only carbon source provided tothe cells, all cell mass produced must have been derived from lactate.

The disclosure of U.S. provisional patent application Ser. No.61/718,024 filed Oct. 24, 2012, is incorporated herein in its entirety.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

The invention claimed is:
 1. A recombinant methanotrophic bacteriumcomprising at least one exogenous nucleic acid encoding an acetateutilization pathway component, wherein acetate is not utilized as acarbon source by an unmodified parent methanotrophic bacterium; whereinthe encoded acetate utilization pathway component comprises anacetyl-CoA synthase and is expressed in a sufficient amount to permitgrowth of the recombinant methanotrophic bacterium on acetate as aprimary carbon source; and wherein the methanotrophic bacterium isselected from Methylococcus capsulatus, Methylomonas sp. 16A,Methylosinus trichosyorium, Methylosinus syorium, Methylocystis parvus,Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus,Methylomonas flagellate, Methylacidiyhilum infernorum, Methylomicrobiumalcaliyhilum, or Methylobacterium organophilum.
 2. The recombinantmethanotrophic bacterium of claim 1, further comprising at least oneexogenous nucleic acid encoding an acetate transporter.
 3. Therecombinant methanotrophic bacterium of claim 1, wherein the parentmethanotrophic bacterium is an obligate methanotrophic bacteriumselected from Methylosinus trichosporium OB3b, Methylococcus capsulatusBath, Methylomnonas sp. 16A, Methylosinus trichosporium, Methylosinussporium, Methylomonas methanica, Methylomonas albus, Methylobactercapsulatus, Methylomonas-sp flasellata AJ-3670, Methylacidiphiluminfernorum, or Methylomicrobium alcaliphilum.
 4. The recombinantmethanotrophic bacterium of claim 1, wherein the parent methanotrophicbacterium is facultative methanotrophic bacterium Methylobacteriumorganophilum.
 5. The recombinant methanotrophic bacterium of claim 1,wherein the at least one exogenous nucleic acid encoding the acetyl-CoAsynthase is codon optimized for the methanotrophic bacterium.
 6. Therecombinant methanotrophic bacterium of claim 2, wherein the exogenousnucleic acid encoding the acetate transporter has at least 80% sequenceidentity with a polynucleotide encoding a functionally active actP fromEscherichia coli, a polynucleotide encoding a functionally active mctPfrom Rhizobium leguminosarum, or a polynucleotide encoding afunctionally active mctC from Corynebacterium glutamicum.
 7. Therecombinant methanotrophic bacterium of claim 2, wherein the encodedacetate transporter comprises a polypeptide having at least 90% aminoacid sequence identity with SEQ ID NO:75.
 8. The recombinantmethanotrophic bacterium of claim 2, wherein the exogenous nucleic acidencoding the acetate transporter comprises or consists of apolynucleotide sequence of SEQ ID NO:113, 118 or
 123. 9. The recombinantmethanotrophic bacterium of claim 2, wherein the exogenous nucleic acidencoding the acetate transporter and the exogenous nucleic acid encodingthe acetyl-CoA synthase are codon optimized for the-host parentmethanotrophic bacterium.
 10. The recombinant methanotrophic bacteriumof claim 9, wherein the parent methanotrophic bacterium is selected fromMethylosinus trichosporium OB3b, Methylococcus capsulatus Bath, orMethylomonas methanica.
 11. The recombinant methanotrophic bacterium ofclaim 1, wherein the exogenous nucleic acid encoding the acetyl-CoAsynthase has at least 80% sequence identity with a polynucleotideencoding a functionally active acsA from Escherichia coil.
 12. Therecombinant methanotrophic bacterium of claim 1, wherein the exogenousnucleic acid encoding the acetyl-CoA synthase comprises or consists of apolynucleotide sequence of SEQ ID NO: 113, 116 or
 121. 13. Therecombinant methanotrophic bacterium of claim 1, wherein the encodedacetyl-CoA synthase comprises a polypeptide having at least 90% sequenceidentity with SEQ ID NO:
 74. 14. The recombinant methanotrophicbacterium of claim 2, wherein the exogenous nucleic acid encoding theacetate transporter and the exogenous nucleic acid encoding theacetyl-CoA synthase are a single nucleic acid molecule.
 15. Therecombinant methanotrophic bacterium of claim 14, wherein the exogenousnucleic acid encoding: (a) the acetate transporter of the single nucleicacid molecule is operably linked to a mdh promoter or a hps promoter:(b) the acetyl-CoA synthase of the single nucleic acid molecule isoperably linked to a mdh promoter or a hps promoter; or (c) the acetatetransporter and the acetyl-CoA synthase comprise an operon in the singlenucleic acid molecule, wherein the operon is operably linked to a mdhpromoter or a hps promoter.
 16. The recombinant methanotrophic bacteriumof claim 15, wherein the mdh promoter comprises or consists of apolynucleotide sequence of SEQ ID NO: 110, and/or the hps promotercomprises or consists of a polynucleotide sequence of SEQ ID NO: 120.17. The recombinant methanotrophic bacterium of claim 14, wherein theexogenous nucleic acid encoding the acetate transporter and theexogenous nucleic acid encoding the acetyl-CoA synthase are present onthe single nucleic acid molecule as an operon.
 18. The recombinantmethanotrophic bacterium of claim 17, wherein the operon encoding theacetate transporter and the acetyl-CoA synthase are codon optimized forthe parent methanotrophic bacterium.
 19. The recombinant methanotrophicbacterium of claim 18, wherein the parent methanotrophic bacterium isMelhylosinus trichosporium OB3b, Methylococcus capsutalus Bath, orMethylomonas methanica.
 20. The recombinant methanotrophic bacterium ofclaim 17, wherein the operon encoding the acetate transporter and theacetyl-CoA synthase comprises or consists of a polynucleotide sequenceof SEQ ID NO: 114, 119 or
 124. 21. A recombinant methanotrophicbacterium, comprising a methanotrophic bacterium containing at least oneexogenous nucleic acid encoding an acetate utilization pathwaycomponent, wherein acetate is not utilized as a carbon source by anunmodified parent methanotrophic bacterium; wherein the encoded acetateutilization pathway component comprises an acetate kinase and isexpressed in a sufficient amount to permit growth of the recombinantmethanotrophic bacterium on acetate as a primary carbon source; andwherein the methanotrophic bacterium is selected from Methylococcuscapsulatus, Methylomonas sp. 16A, Methylosinus trichosporium,Methylosinus sporium, Methylocystis parvus, Methylomonas methanica,Methylomonas albus, Methylobacter capsulatus, Methylomonas flagellata,Methylacidiphilum infernorum, Methylomicrobium alcaliphilum, orMethylobacterium organophilum.
 22. The recombinant methanotrophicbacterium of claim 21, further comprising at least one exogenous nucleicacid encoding a phosphotransacetylase.
 23. The recombinantmethanotrophic bacterium of claim 21, further comprising at least oneexogenous nucleic acid encoding an acetate transporter.
 24. Therecombinant methanotrophic bacterium of claim 21, wherein the parentmethanotrophic bacterium is an obligate methanotrophic bacteriumselected from Methylosinus trichosporium OB3b, Methylococcus capsulatusBath, Methylomonas sp. 16A, Methylosinus trichosporium, Methylosinussporium, Methylomonas methanica, Methylomonas albus, Methylobactercapsulatus, Methylomonas flagellata AJ-3670, Methylacidiphiluminfemorum, or Methylomicrobium alcaliphilum.
 25. The recombinantmethanotrophic bacterium of claim 21, wherein the exogenous nucleic acidencoding the acetate kinase is codon optimized for the parentmethanotrophic bacterium.
 26. The recombinant methanotrophic bacteriumof claim 22, wherein the exogenous nucleic acid encoding the acetatekinase and the exogenous nucleic acid encoding the phosphotransacetylaseare codon optimized for the parent methanotrophic bacterium.
 27. Therecombinant methanotrophic bacterium of claim 26, wherein the parentmethanotrophic bacterium is selected from Methylosinus trichosporiumOB3b, Methylococcus capsulatus Bath, or Methylomonas methanica.
 28. Therecombinant methanotrophic bacterium of claim 26, wherein the at leastone exogenous nucleic acid encodes an acetate transporter, wherein theacetate transporter encoding nucleic acid is codon optimized for theparent methanotrophic bacterium.
 29. The recombinant methanotrophicbacterium of claim 22, wherein the exogenous nucleic acid encoding theacetate kinase and the exogenous nucleic acid encoding thephosphotransacetylase are a single nucleic acid molecule.
 30. Therecombinant methanotrophic bacterium of claim 29, wherein the exogenousnucleic acid encoding: (a) the acetate kinase of the single nucleic acidmolecule is operably linked to a mdh promoter or a hps promoter; (b) thephosphotransacetylase of the single nucleic acid molecule is operablylinked to a mdh promoter or a hps promoter; or (c) the acetate kinaseand the phosphotransacetylase comprise an operon in the single nucleicacid molecule, wherein the operon is operably linked to a mdh promoteror a hps promoter.
 31. The recombinant methanotrophic bacterium of claim24, wherein the mdh promoter comprises or consists of a polynucleotidesequence of SEQ ID NO:110, and/or the hps promoter comprises or consistsof a polynucleotide sequence of SEQ ID NO:120.
 32. The recombinantmethanotrophic bacterium of claim 29, wherein the exogenous nucleic acidencoding the acetate kinase and the exogenous nucleic acid encoding thephosphotransacetylase are present on the single nucleic acid molecule asan operon.
 33. The recombinant methanotrophic bacterium of claim 32,wherein the operon encoding the acetate kinase and thephosphotransacetylase are codon optimized for the parent methanotrophicbacterium.
 34. The recombinant methanotrophic bacterium of claim 32,wherein the parent methanotrophic bacterium is selected fromMethylosinus trichosporium OB3b, Methylococcus capsulatus Bath, orMethylomonas methanica.